AAV Vectors Targeted to the Central Nervous System

ABSTRACT

The invention relates to chimeric AAV capsids targeted to the central nervous system, virus vectors comprising the same, and methods of using the vectors to target the central nervous system. The invention further relates to chimeric AAV capsids targeted to oligodendrocytes, virus vectors comprising the same, and methods of using the vectors to target oligodendrocytes.

STATEMENT OF PRIORITY

This application is a continuation of and claims priority to U.S. patentapplication Ser. No. 15/525,214, filed May 8, 2017, which is a 35 U.S.C.§ 371 national phase application of PCT/US2015/061788, filed Nov. 20,2015, which claims the benefit of U.S. Provisional Patent ApplicationSer. No. 62/082,897, filed Nov. 21, 2014 and U.S. Provisional PatentApplication Ser. No. 62/218,857, filed Sep. 15, 2015, the entirecontents of each of which are incorporated by reference herein.

STATEMENT REGARDING ELECTRONIC FILING OF A SEQUENCE LISTING

A Sequence Listing in ASCII text format, submitted under 37 C.F.R. §1.821, entitled 5470-718CT2 ST25.txt, 638,580 bytes in size, generatedon Jan. 13, 2020 and filed via EFS-Web, is provided in lieu of a papercopy. This Sequence Listing is hereby incorporated by reference into thespecification for its disclosures.

FIELD OF THE INVENTION

The invention relates to chimeric AAV capsids targeted to the centralnervous system, virus vectors comprising the same, and methods of usingthe vectors to target the central nervous system. The invention furtherrelates to chimeric AAV capsids targeted to oligodendrocytes, virusvectors comprising the same, and methods of using the vectors to targetoligodendrocytes.

BACKGROUND OF THE INVENTION

Adeno-associated virus (AAV) was first reported to efficiently transducemuscle over ten years ago (Xiao et al., (1996) J. Virol. 70:8098-8108).The recombinant AAV (rAAV) genome composed of a foreign expressioncassette and AAV inverted terminal repeat (ITR) sequences exists ineukaryotic cells in an episomal form that is responsible for persistenttransgene expression (Schnepp et al., (2003) J. Virol. 77:3495-3504).AAV vectors have a good safety profile. No human disease has beenassociated with wild-type AAV infection and low toxicity is observed inhuman subjects following transduction by rAAV (Manno et al., (2003)Blood 101:2963-2972).

AAV vectors have been used in clinical trials for central nervous system(CNS) disorders. While some success have been garnered,naturally-occurring AAV capsids lack specificity for the CNS and areunsuitable for certain disease applications. Recent advances in AAVengineering and directed evolution have expanded the ability to developnovel AAV serotypes, including vectors with altered tropism (Gray etal., (2010) Mol. Ther. 18:570-578). However, no AAV vectors have beencapable of widespread CNS gene transfer with minimal tropism forperipheral organs.

In the brain, the vast majority of AAV vectors exhibit a dominantpreference for neurons with a very low efficacy for other cell types,such as oligodendrocytes. AAV vectors that efficiently targetoligodendrocytes have not been developed.

SUMMARY OF THE INVENTION

The present invention is based, in part, on the development of chimericAAV capsid sequences that are capable of widespread CNS gene transferafter delivery to the CNS with minimal tropism for peripheral organs.The invention further relates to chimeric AAV capsids that have enhancedtransduction capabilities in subjects with Rett Syndrome. The chimericcapsids can be used to create AAV vectors for use in research ortherapeutic applications where widespread CNS gene transfer is desiredwithout extensive vector biodistribution to peripheral organs.

The present invention further is based, in part, on the development ofchimeric AAV capsid sequences that are capable ofoligodendrocyte-preferred or specific gene transfer after delivery tothe CNS with minimal tropism for peripheral organs. The chimeric capsidscan be used to create AAV vectors for use in research or therapeuticapplications where oligodendrocyte gene transfer is desired withoutextensive vector biodistribution to neurons or to peripheral organs.

Thus, one aspect of the invention relates to a nucleic acid encoding anAAV capsid, the nucleic acid comprising an AAV capsid coding sequencethat is at least 70% identical to: (a) the nucleotide sequence of anyone of SEQ ID NOS:1-43; or (b) a nucleotide sequence encoding any one ofSEQ ID NOS:44-86, along with cells and viral particles comprising thenucleic acid.

Thus, one aspect of the invention relates to a nucleic acid encoding anAAV capsid, the nucleic acid comprising an AAV capsid coding sequencethat is at least 70% identical to: (a) the nucleotide sequence of anyone of SEQ ID NOS: 87-107; or (b) a nucleotide sequence encoding any oneof SEQ ID NOS: 108-128, along with cells and viral particles comprisingthe nucleic acid.

Another aspect of the invention relates to an AAV capsid comprising anamino acid sequence at least 90% identical to any one of SEQ IDNOS:44-86, along with AAV particles comprising an AAV vector genome andthe AAV capsid of the invention.

Another aspect of the invention relates to an AAV capsid comprising anamino acid sequence at least 90% identical to any one of SEQ ID NOS:108-128, along with AAV particles comprising an AAV vector genome andthe AAV capsid of the invention.

A further aspect of the invention relates to a method of producing arecombinant AAV particle comprising an AAV capsid, the methodcomprising: providing a cell in vitro with a nucleic acid of theinvention, an AAV rep coding sequence, an AAV vector genome comprising aheterologous nucleic acid, and helper functions for generating aproductive AAV infection; and allowing assembly of the recombinant AAVparticle comprising the AAV capsid and encapsidating the AAV vectorgenome.

An additional aspect of the invention relates to a pharmaceuticalformulation comprising the nucleic acid, virus particle, AAV capsid, orAAV particle of the invention in a pharmaceutically acceptable carrier.

Another aspect of the invention relates to a method of delivering anucleic acid of interest to a CNS cell, the method comprising contactingthe cell with the AAV particle of the invention.

A further aspect of the invention relates to a method of delivering anucleic acid of interest to a CNS cell in a mammalian subject, themethod comprising administering an effective amount of the AAV particleor pharmaceutical formulation of the invention to a mammalian subject.

An additional aspect of the invention relates to a method of deliveringa nucleic acid of interest to an area of the CNS bordering a compromisedblood brain barrier area in a mammalian subject, the method comprisingintravenously administering an effective amount of the AAV particle orpharmaceutical formulation of the invention to a mammalian subject.

Another aspect of the invention relates to a method of treating adisorder associated with CNS dysfunction in a mammalian subject in needthereof, the method comprising administering a therapeutically effectiveamount of the AAV particle or pharmaceutical formulation of theinvention to a mammalian subject.

Another aspect of the invention relates to a method of treating RettSyndrome in a mammalian subject in need thereof, the method comprisingadministering a therapeutically effective amount of the AAV particle orpharmaceutical formulation of the invention to a mammalian subject.

A further aspect of the invention relates to a method of preparing anAAV capsid having a tropism profile of interest, the method comprisingmodifying the AAV capsid of the invention to insert an amino acidsequence providing the tropism profile of interest.

One aspect of the invention relates to a nucleic acid encoding an AAVcapsid, the nucleic acid comprising an AAV capsid coding sequence thatis at least 70% identical to: (a) the nucleotide sequence of any one ofSEQ ID NO:129; or (b) a nucleotide sequence encoding any one of SEQ IDNOS:130-132, along with cells and viral particles comprising the nucleicacid.

Another aspect of the invention relates to an AAV capsid comprising anamino acid sequence at least 90% identical to any one of SEQ IDNOS:130-132, along with AAV particles comprising an AAV vector genomeand the AAV capsid of the invention.

Another aspect of the invention relates to a method of delivering anucleic acid of interest to an oligodendrocyte, the method comprisingcontacting the cell with the AAV particle of the invention.

A further aspect of the invention relates to a method of delivering anucleic acid of interest to an oligodendrocyte in a mammalian subject,the method comprising administering an effective amount of the AAVparticle or pharmaceutical formulation of the invention to a mammaliansubject.

Another aspect of the invention relates to a method of treating adisorder associated with oligodendrocyte dysfunction in a mammaliansubject in need thereof, the method comprising administering atherapeutically effective amount of the AAV particle or pharmaceuticalformulation of the invention to a mammalian subject.

A further aspect of the invention relates to a nucleic acid encoding anAAV8 capsid comprising an E532K substitution, along with cells and viralparticles comprising the nucleic acid.

Another aspect of the invention relates to an AAV8 capsid comprising anE532K substitution, along with AAV particles comprising an AAV vectorgenome and the AAV capsid of the invention.

Another aspect of the invention relates to a method of delivering anucleic acid of interest to an oligodendrocyte, the method comprisingcontacting the cell with the AAV particle of the invention.

A further aspect of the invention relates to a method of delivering anucleic acid of interest to an oligodendrocyte in a mammalian subject,the method comprising administering an effective amount of the AAVparticle or pharmaceutical formulation of the invention to a mammaliansubject.

Another aspect of the invention relates to a method of treating adisorder associated with oligodendrocyte dysfunction in a mammaliansubject in need thereof, the method comprising administering atherapeutically effective amount of the AAV particle or pharmaceuticalformulation of the invention to a mammalian subject.

These and other aspects of the invention are set forth in more detail inthe description of the invention below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the chimeric structure of AAV capsid clones isolated fromthe spinal cord of wild-type mice.

FIG. 2 shows the chimeric structure of AAV capsid clones isolated fromthe brain of wild-type mice.

FIG. 3 shows the chimeric structure of AAV capsid clones isolated fromRett Syndrome mice.

FIGS. 4A-4E show the tropism of isolated clones.

FIG. 5 shows the tropism of isolated clones.

FIG. 6 shows the tropism of isolated clones.

FIG. 7 shows the transduction efficiency of isolated clones.

FIG. 8 shows the MeCP2 tropism of isolated clones.

FIG. 9 shows the NeuN tropism of isolated clones.

FIGS. 10A-10E shows that Olig001 has an oligodendrocyte preferringtropism. FIG. 10A: Diagram of the cap gene from Olig001 compared toAAV8. The different colors represent a different AAV parental serotype(blue=AAV2, purple=AAV8, red=AAV9, yellow=AAV1, and orange=AAV6) presentin the input reaction of the library. The black vertical bars indicatepoint mutations. FIG. 10B: Olig001 transduction of cells in the ratstriatum that exhibit characteristics indicative of oligodendrocytesincluding the localization of GFP positive myelin in the patch of thestriatal patch/matrix. FIG. 10C: Confocal image at a highermagnification of the transduced cells that again reflects the uniquemorphology of CNS oligodendroctyes. FIG. 10D: Confocal image reveals alack of co-localization of the GFP positive cells with astrocyteslabeled with GFAP (red) within the striatum. FIG. 10E: Confocal imageillustrates that the vast majority of Olig001 transduced cells withinthe striatum do not co-localize with a marker of neurons, NeuN (red).However, the arrow in indicates a single GFP/NeuN positive cell.

FIG. 11 shows Olig001 is detargeted from peripheral tissues compared toAAV8. Adult female C57Bl/6 mice received an intravenous dose of 5×10¹⁰vg (˜2.5×10¹² vg/kg body weight) of either Olig001-CBh-GFP (white bars;n=4) or AAV8-CBh-GFP (gray bars; n=5). Ten days later the organdistribution of GFP genome per diploid mouse genome (LaminB2) wasdetermined by qPCR. Error bars indicate standard error of the mean.

FIGS. 12A-12G shows that AAV8 with an E532K mutation is oligotropic.FIG. 12A: Diagram of the cap gene from Olig001 compared to AAV8/E532K.The different colors represent a different AAV parental serotype(blue=AAV2, purple=AAV8, red=AAV9, yellow=AAV1, and orange=AAV6). Theblack vertical bars indicate point mutations. AAV8/E532K was packagedwith CBh-GFP, and 2×10⁸ vg was intracranially injected into the striatumof wild-type male Sprague-Dawley rats. Two weeks post injection the ratswere transcardially perfused and their brains fixed and sectionedcoronally. FIGS. 12B-12D: Confocal images of the striatum indicate thatGFP positive cells lack co-localization with neuronal (NeuN) markers.FIGS. 12E-12G: Confocal images of the striatum indicate that GFPpositive cells lack co-localization and astrocyte (GFAP) marker.

FIGS. 13A-13G show that Olig001 oligodendrocyte preferring tropism isindependent of VP3 sequence. FIG. 13A: Diagram of the cap gene fromOlig001 compared to Olig001/AAV VP3. The different colors represent adifferent AAV parental serotype (blue=AAV2, purple=AAV8, red=AAV9,yellow=AAV1, and orange=AAV6). The black vertical bars indicate pointmutations. Mutant Olig001 with VP3 of AAV8 (Olig001/AAV8 VP3) waspackaged with CBh-GFP at a titer of 2×10⁸ vg/μl and intracraniallyinjected into the striatum of wild-type male Sprague-Dawley rats. Twoweeks later the rats were transcardially perfused and their brains fixedand sectioned coronally. FIGS. 13B-13D: Confocal images of the striatumindicate that GFP positive cells exhibit striatal oligodendrocytemorphology and lack co-localization with neuronal (NeuN) markers. FIGS.13E-13G: Confocal images of the striatum indicate that GFP positivecells exhibit striatal oligodendrocyte morphology and lackco-localization and astrocyte (GFAP) marker.

FIGS. 14A-14B shows that in vitro binding analysis agrees with in vivotropism. In vitro mixed glia cultures were created by dissociatingneonatal day 3 mouse brains. Cultures were incubated with an equivalentamount of either AAV8-CBh-GFP or Olig001-CBh-GFP for 1 h at 4° C. toallow for vector binding, but not uptake. FIG. 14A: The amount of vectorbound to cells was quantified by qPCR for GFP and normalized to mousegenomic LaminB2. Error bars indicate Standard Error of the Mean, *indicates a significant difference of P<0.03, and ** indicates asignificant difference of P<0.01. FIG. 14B: The fold difference wasdetermined using the average binding for each virus compared to AAV8.

FIG. 15 shows a summary of the findings. Diagrams of the cap genes usedin this study with the in vivo dominant tropism when infused into theadult rat striatum and in vitro binding fold over AAV8. The differentcolors represent a different AAV parental serotype (blue=AAV2,purple=AAV8, red=AAV9, yellow=AAV1, and orange=AAV6). The black verticalbars indicate point mutations. ND=not determined and * indicates datanot shown.

FIG. 16 shows mutants derived from Olig001 are detargeted fromperipheral tissues compared to AAV8 (see also FIG. 11). Adult femaleC57Bl/6 mice received an intravenous dose of 5×10¹⁰ vg (˜2.5×10¹² vg/kgbody weight) of either Olig001 or one of its mutant derivatives, asindicated, packaging the sc CBh-GFP genome. Ten days later the organdistribution of GFP genome per diploid mouse genome (LaminB2) wasdetermined by qPCR. Error bars indicate standard error of the mean.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based, in part, on the development of chimericAAV capsid sequences that are capable of widespread CNS gene transferafter delivery to the CNS with minimal tropism for peripheral organs.The invention further relates to chimeric AAV capsids that have enhancedtransduction capabilities in subjects with Rett Syndrome. The chimericcapsids can be used to create AAV vectors for use in research ortherapeutic applications where widespread CNS gene transfer is desiredwithout extensive vector biodistribution to peripheral organs.

The present invention further is based, in part, on the development ofchimeric AAV capsid sequences that are capable ofoligodendrocyte-preferred or specific gene transfer after delivery tothe CNS with minimal tropism for peripheral organs. The chimeric capsidscan be used to create AAV vectors for use in research or therapeuticapplications where oligodendrocyte gene transfer is desired withoutextensive vector biodistribution to neurons or to peripheral organs.

The present invention is explained in greater detail below. Thisdescription is not intended to be a detailed catalog of all thedifferent ways in which the invention may be implemented, or all thefeatures that may be added to the instant invention. For example,features illustrated with respect to one embodiment may be incorporatedinto other embodiments, and features illustrated with respect to aparticular embodiment may be deleted from that embodiment. In addition,numerous variations and additions to the various embodiments suggestedherein will be apparent to those skilled in the art in light of theinstant disclosure which do not depart from the instant invention.Hence, the following specification is intended to illustrate someparticular embodiments of the invention, and not to exhaustively specifyall permutations, combinations and variations thereof.

Unless the context indicates otherwise, it is specifically intended thatthe various features of the invention described herein can be used inany combination. Moreover, the present invention also contemplates thatin some embodiments of the invention, any feature or combination offeatures set forth herein can be excluded or omitted. To illustrate, ifthe specification states that a complex comprises components A, B and C,it is specifically intended that any of A, B or C, or a combinationthereof, can be omitted and disclaimed singularly or in any combination.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. The terminology used in thedescription of the invention herein is for the purpose of describingparticular embodiments only and is not intended to be limiting of theinvention.

Nucleotide sequences are presented herein by single strand only, in the5′ to 3′ direction, from left to right, unless specifically indicatedotherwise. Nucleotides and amino acids are represented herein in themanner recommended by the IUPAC-IUB Biochemical Nomenclature Commission,or (for amino acids) by either the one-letter code, or the three lettercode, both in accordance with 37 C.F.R. § 1.822 and established usage.

Except as otherwise indicated, standard methods known to those skilledin the art may be used for production of recombinant and syntheticpolypeptides, antibodies or antigen-binding fragments thereof,manipulation of nucleic acid sequences, production of transformed cells,the construction of rAAV constructs, modified capsid proteins, packagingvectors expressing the AAV rep and/or cap sequences, and transiently andstably transfected packaging cells. Such techniques are known to thoseskilled in the art. See, e.g., SAMBROOK et al., MOLECULAR CLONING: ALABORATORY MANUAL 2nd Ed. (Cold Spring Harbor, N.Y., 1989); F. M.AUSUBEL et al. CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Green PublishingAssociates, Inc. and John Wiley & Sons, Inc., New York).

All publications, patent applications, patents, nucleotide sequences,amino acid sequences and other references mentioned herein areincorporated by reference in their entirety.

I. Definitions

The designation of all amino acid positions in the AAV capsid subunitsin the description of the invention and the appended claims is withrespect to VP1 capsid subunit numbering.

As used in the description of the invention and the appended claims, thesingular forms “a,” “an” and “the” are intended to include the pluralforms as well, unless the context clearly indicates otherwise.

As used herein, “and/or” refers to and encompasses any and all possiblecombinations of one or more of the associated listed items, as well asthe lack of combinations when interpreted in the alternative (“or”).

Moreover, the present invention also contemplates that in someembodiments of the invention, any feature or combination of features setforth herein can be excluded or omitted.

Furthermore, the term “about,” as used herein when referring to ameasurable value such as an amount of a compound or agent of thisinvention, dose, time, temperature, and the like, is meant to encompassvariations of ±20%, ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of thespecified amount.

The term “consisting essentially of” as used herein in connection with anucleic acid, protein or capsid structure means that the nucleic acid,protein or capsid structure does not contain any element other than therecited element(s) that significantly alters (e.g., more than about 1%,5% or 10%) the function of interest of the nucleic acid, protein orcapsid structure, e.g., tropism profile of the protein or capsid or aprotein or capsid encoded by the nucleic acid.

The term “adeno-associated virus” (AAV) in the context of the presentinvention includes without limitation AAV type 1, AAV type 2, AAV type 3(including types 3A and 3B), AAV type 4, AAV type 5, AAV type 6, AAVtype 7, AAV type 8, AAV type 9, AAV type 10, AAV type 11, avian AAV,bovine AAV, canine AAV, equine AAV, and ovine AAV and any other AAV nowknown or later discovered. See, e.g., BERNARD N. FIELDS et al.,VIROLOGY, volume 2, chapter 69 (4th ed., Lippincott-Raven Publishers). Anumber of additional AAV serotypes and clades have been identified (see,e.g., Gao et al., (2004) J. Virol. 78:6381-6388 and Table 1), which arealso encompassed by the term “AAV.”

The genomic sequences of various AAV and autonomous parvoviruses, aswell as the sequences of the ITRs, Rep proteins, and capsid subunits areknown in the art. Such sequences may be found in the literature or inpublic databases such as the GenBank® database. See, e.g., GenBank®Accession Numbers NC 002077, NC 001401, NC 001729, NC 001863, NC 001829,NC 001862, NC 000883, NC 001701, NC 001510, AF063497, U89790, AF043303,AF028705, AF028704, J02275, J01901, J02275, X01457, AF288061, AH009962,AY028226, AY028223, NC 001358, NC 001540, AF513851, AF513852, AY530579,AY631965, AY631966; the disclosures of which are incorporated herein intheir entirety. See also, e.g., Srivistava et al., (1983) J. Virol.45:555; Chiorini et al., (1998) J. Virol. 71:6823; Chiorini et al.,(1999) J. Virol. 73:1309; Bantel-Schaal et al., (1999) J. Virol. 73:939;Xiao et al., (1999) J. Virol. 73:3994; Muramatsu et al., (1996) Virology221:208; Shade et al., (1986) J. Virol. 58:921; Gao et al., (2002) Proc.Nat. Acad. Sci. USA 99:11854; international patent publications WO00/28061, WO 99/61601, WO 98/11244; U.S. Pat. No. 6,156,303; thedisclosures of which are incorporated herein in their entirety. See alsoTable 1. An early description of the AAV1, AAV2 and AAV3 terminal repeatsequences is provided by Xiao, X., (1996), “Characterization ofAdeno-associated virus (AAV) DNA replication and integration,” Ph.D.Dissertation, University of Pittsburgh, Pittsburgh, Pa. (incorporatedherein it its entirety).

TABLE 1 Complete Genomes GenBank Accession Number Adeno-associated virus1 NC_002077, AF063497 Adeno-associated virus 2 NC_001401Adeno-associated virus 3 NC_001729 Adeno-associated virus 3B NC_001863Adeno-associated virus 4 NC_001829 Adeno-associated virus 5 Y18065,AF085716 Adeno-associated virus 6 NC_001862 Avian AAV ATCC VR-865AY186198, AY629583, NC_004828 Avian AAV strain DA-1 NC_006263, AY629583Bovine AAV NC_005889, AY388617 Clade A AAV1 NC_002077, AF063497 AAV6NC_001862 Hu.48 AY530611 Hu 43 AY530606 Hu 44 AY530607 Hu 46 AY530609Clade B Hu. 19 AY530584 Hu. 20 AY530586 Hu 23 AY530589 Hu22 AY530588Hu24 AY530590 Hu21 AY530587 Hu27 AY530592 Hu28 AY530593 Hu 29 AY530594Hu63 AY530624 Hu64 AY530625 Hu13 AY530578 Hu56 AY530618 Hu57 AY530619Hu49 AY530612 Hu58 AY530620 Hu34 AY530598 Hu35 AY530599 AAV2 NC_001401Hu45 AY530608 Hu47 AY530610 Hu51 AY530613 Hu52 AY530614 Hu T41 AY695378Hu S17 AY695376 Hu T88 AY695375 Hu T71 AY695374 Hu T70 AY695373 Hu T40AY695372 Hu T32 AY695371 Hu T17 AY695370 Hu LG15 AY695377 Clade C Hu9AY530629 Hu10 AY530576 Hu11 AY530577 Hu53 AY530615 Hu55 AY530617 Hu54AY530616 Hu7 AY530628 Hu18 AY530583 Hu15 AY530580 Hu16 AY530581 Hu25AY530591 Hu60 AY530622 Ch5 AY243021 Hu3 AY530595 Hu1 AY530575 Hu4AY530602 Hu2 AY530585 Hu61 AY530623 Clade D Rh62 AY530573 Rh48 AY530561Rh54 AY530567 Rh55 AY530568 Cy2 AY243020 AAV7 AF513851 Rh35 AY243000Rh37 AY242998 Rh36 AY242999 Cy6 AY243016 Cy4 AY243018 Cy3 AY243019 Cy5AY243017 Rh13 AY243013 Clade E Rh38 AY530558 Hu66 AY530626 Hu42 AY530605Hu67 AY530627 Hu40 AY530603 Hu41 AY530604 Hu37 AY530600 Rh40 AY530559Rh2 AY243007 Bb1 AY243023 Bb2 AY243022 Rh10 AY243015 Hu17 AY530582 Hu6AY530621 Rh25 AY530557 Pi2 AY530554 Pi1 AY530553 Pi3 AY530555 Rh57AY530569 Rh50 AY530563 Rh49 AY530562 Hu39 AY530601 Rh58 AY530570 Rh61AY530572 Rh52 AY530565 Rh53 AY530566 Rh51 AY530564 Rh64 AY530574 Rh43AY530560 AAV8 AF513852 Rh8 AY242997 Rh1 AY530556 Clade F Hu14 (AAV9)AY530579 Hu31 AY530596 Hu32 AY530597 Clonal Isolate AAV5 Y18065,AF085716 AAV 3 NC_001729 AAV 3B NC_001863 AAV4 NC_001829 Rh34 AY243001Rh33 AY243002 Rh32 AY243003

A “chimeric” AAV nucleic acid capsid coding sequence or AAV capsidprotein is one that combines portions of two or more capsid sequences. A“chimeric” AAV virion or particle comprises a chimeric AAV capsidprotein.

The term “tropism” as used herein refers to preferential entry of thevirus into certain cell or tissue type(s) and/or preferentialinteraction with the cell surface that facilitates entry into certaincell or tissue types, optionally and preferably followed by expression(e.g., transcription and, optionally, translation) of sequences carriedby the viral genome in the cell, e.g., for a recombinant virus,expression of the heterologous nucleotide sequence(s). Those skilled inthe art will appreciate that transcription of a heterologous nucleicacid sequence from the viral genome may not be initiated in the absenceof trans-acting factors, e.g., for an inducible promoter or otherwiseregulated nucleic acid sequence. In the case of a rAAV genome, geneexpression from the viral genome may be from a stably integratedprovirus and/or from a non-integrated episome, as well as any other formwhich the virus nucleic acid may take within the cell.

The term “tropism profile” refers to the pattern of transduction of oneor more target cells, tissues and/or organs. Representative examples ofchimeric AAV capsids have a tropism profile characterized by efficienttransduction of cells of the CNS with only low transduction ofperipheral organs.

The term “specific for cells of the CNS” as used herein refers to aviral vector that, when administered directly into the CNS,preferentially transduces all cell types in the CNS with minimaltransduction of cells outside the CNS. In some embodiments, at leastabout 80% of the transduced cells are CNS cells, e.g., at least about85%, 90%, 95%, 96%, 97%, 98%, 99% or more CNS cells.

The term “disorder associated with CNS dysfunction” as used hereinrefers to a disease, disorder, or injury in which CNS cells are damaged,lost, or function improperly. The term includes diseases, disorders, andinjuries in which CNS cells are directly affected as well as diseases,disorders, and injuries in which CNS cells become dysfunctionalsecondary to damage to other cells (e.g., myocardial infarction orstroke).

The term “specific for oligodendrocytes” as used herein refers to aviral vector that, when administered directly into the CNS,preferentially transduces oligodendrocytes over neurons, astrocytes, andother CNS cell types. In some embodiments, at least about 80% of thetransduced cells are oligodendrocytes, e.g., at least about 85%, 90%,95%, 96%, 97%, 98%, 99% or more oligodendrocytes.

The term “disorder associated with oligodendrocyte dysfunction” as usedherein refers to a disease, disorder, or injury in whicholigodendrocytes are damaged, lost, or function improperly. The termincludes diseases, disorders, and injuries in which oligodendrocytes aredirectly affected as well as diseases, disorders, and injuries in whicholigodendrocytes become dysfunctional secondary to damage to other cells(e.g., spinal cord injury).

The term “bordering a compromised blood-brain barrier area” as usedherein refers to CNS cells that are adjacent to a portion of theblood-brain barrier in which the barrier function has been compromised.

As used herein, “transduction” of a cell by a virus vector (e.g., an AAVvector) means entry of the vector into the cell and transfer of geneticmaterial into the cell by the incorporation of nucleic acid into thevirus vector and subsequent transfer into the cell via the virus vector.

Unless indicated otherwise, “efficient transduction” or “efficienttropism,” or similar terms, can be determined by reference to a suitablepositive or negative control (e.g., at least about 50%, 60%, 70%, 80%,85%, 90%, 95% or more of the transduction or tropism, respectively, of apositive control or at least about 110%, 120%, 150%, 200%, 300%, 500%,1000% or more of the transduction or tropism, respectively, of anegative control).

Similarly, it can be determined if a virus “does not efficientlytransduce” or “does not have efficient tropism” for a target tissue, orsimilar terms, by reference to a suitable control. In particularembodiments, the virus vector does not efficiently transduce (i.e., doesnot have efficient tropism for) tissues outside the CNS, e.g., liver,kidney, gonads and/or germ cells. In particular embodiments, undesirabletransduction of tissue(s) (e.g., liver) is 20% or less, 10% or less, 5%or less, 1% or less, 0.1% or less of the level of transduction of thedesired target tissue(s) (e.g., CNS cells).

As used herein, the term “polypeptide” encompasses both peptides andproteins, unless indicated otherwise.

A “nucleic acid” or “nucleotide sequence” is a sequence of nucleotidebases, and may be RNA, DNA or DNA-RNA hybrid sequences (including bothnaturally occurring and non-naturally occurring nucleotide), but ispreferably either single or double stranded DNA sequences.

As used herein, an “isolated” nucleic acid or nucleotide sequence (e.g.,an “isolated DNA” or an “isolated RNA”) means a nucleic acid ornucleotide sequence separated or substantially free from at least someof the other components of the naturally occurring organism or virus,for example, the cell or viral structural components or otherpolypeptides or nucleic acids commonly found associated with the nucleicacid or nucleotide sequence.

Likewise, an “isolated” polypeptide means a polypeptide that isseparated or substantially free from at least some of the othercomponents of the naturally occurring organism or virus, for example,the cell or viral structural components or other polypeptides or nucleicacids commonly found associated with the polypeptide.

By the term “treat,” “treating,” or “treatment of” (or grammaticallyequivalent terms) it is meant that the severity of the subject'scondition is reduced or at least partially improved or amelioratedand/or that some alleviation, mitigation or decrease in at least oneclinical symptom is achieved and/or there is a delay in the progressionof the condition and/or prevention or delay of the onset of a disease ordisorder.

As used herein, the term “prevent,” “prevents,” or “prevention” (andgrammatical equivalents thereof) refers to a delay in the onset of adisease or disorder or the lessening of symptoms upon onset of thedisease or disorder. The terms are not meant to imply complete abolitionof disease and encompasses any type of prophylactic treatment thatreduces the incidence of the condition or delays the onset and/orprogression of the condition.

An “effective” or “therapeutically effective” amount as used herein isan amount that is sufficient to provide some improvement or benefit tothe subject. Alternatively stated, an “effective” or “therapeuticallyeffective” amount is an amount that will provide some alleviation,mitigation, or decrease in at least one clinical symptom in the subject.Those skilled in the art will appreciate that the therapeutic effectsneed not be complete or curative, as long as some benefit is provided tothe subject.

A “heterologous nucleotide sequence” or “heterologous nucleic acid” is asequence that is not naturally occurring in the virus. Generally, theheterologous nucleic acid or nucleotide sequence comprises an openreading frame that encodes a polypeptide and/or a nontranslated RNA.

A “therapeutic polypeptide” can be a polypeptide that can alleviate orreduce symptoms that result from an absence or defect in a protein in acell or subject. In addition, a “therapeutic polypeptide” can be apolypeptide that otherwise confers a benefit to a subject, e.g.,anti-cancer effects or improvement in transplant survivability.

As used herein, the term “vector,” “virus vector,” “delivery vector”(and similar terms) generally refers to a virus particle that functionsas a nucleic acid delivery vehicle, and which comprises the viralnucleic acid (i.e., the vector genome) packaged within the virion. Virusvectors according to the present invention comprise a chimeric AAVcapsid according to the invention and can package an AAV or rAAV genomeor any other nucleic acid including viral nucleic acids. Alternatively,in some contexts, the term “vector,” “virus vector,” “delivery vector”(and similar terms) may be used to refer to the vector genome (e.g.,vDNA) in the absence of the virion and/or to a viral capsid that acts asa transporter to deliver molecules tethered to the capsid or packagedwithin the capsid.

A “recombinant AAV vector genome” or “rAAV genome” is an AAV genome(i.e., vDNA) that comprises at least one inverted terminal repeat (e.g.,one, two or three inverted terminal repeats) and one or moreheterologous nucleotide sequences. rAAV vectors generally retain the 145base terminal repeat(s) (TR(s)) in cis to generate virus; however,modified AAV TRs and non-AAV TRs including partially or completelysynthetic sequences can also serve this purpose. All other viralsequences are dispensable and may be supplied in trans (Muzyczka, (1992)Curr. Topics Microbiol. Immunol. 158:97). The rAAV vector optionallycomprises two TRs (e.g., AAV TRs), which generally will be at the 5′ and3′ ends of the heterologous nucleotide sequence(s), but need not becontiguous thereto. The TRs can be the same or different from eachother. The vector genome can also contain a single ITR at its 3′ or 5′end.

The term “terminal repeat” or “TR” includes any viral terminal repeat orsynthetic sequence that forms a hairpin structure and functions as aninverted terminal repeat (i.e., mediates the desired functions such asreplication, virus packaging, integration and/or provirus rescue, andthe like). The TR can be an AAV TR or a non-AAV TR. For example, anon-AAV TR sequence such as those of other parvoviruses (e.g., canineparvovirus (CPV), mouse parvovirus (MVM), human parvovirus B-19) or theSV40 hairpin that serves as the origin of SV40 replication can be usedas a TR, which can further be modified by truncation, substitution,deletion, insertion and/or addition. Further, the TR can be partially orcompletely synthetic, such as the “double-D sequence” as described inU.S. Pat. No. 5,478,745 to Samulski et al.

An “AAV terminal repeat” or “AAV TR” may be from any AAV, including butnot limited to serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 or anyother AAV now known or later discovered (see, e.g., Table 1). An AAVterminal repeat need not have the native terminal repeat sequence (e.g.,a native AAV TR sequence may be altered by insertion, deletion,truncation and/or missense mutations), as long as the terminal repeatmediates the desired functions, e.g., replication, virus packaging,integration, and/or provirus rescue, and the like.

The terms “rAAV particle” and “rAAV virion” are used interchangeablyhere. A “rAAV particle” or “rAAV virion” comprises a rAAV vector genomepackaged within an AAV capsid.

The AAV capsid structure is described in more detail in BERNARD N.FIELDS et al., VIROLOGY, volume 2, chapters 69 & 70 (4th ed.,Lippincott-Raven Publishers).

By “substantially retain” a property, it is meant that at least about75%, 85%, 90%, 95%, 97%, 98%, 99% or 100% of the property (e.g.,activity or other measurable characteristic) is retained.

II. Chimeric AAV Capsids Targeted to the CNS

The inventors have identified chimeric AAV capsid structures capable ofproviding widespread CNS gene transfer with minimal tropism forperipheral organs. Thus, one aspect of the invention relates to chimericAAV capsid structures capable of providing CNS gene transfer in asubject, e.g., a wild-type subject, e.g., a subject that does not have aCNS disorder. In certain embodiments, the invention relates to a nucleicacid encoding an AAV capsid, the nucleic acid comprising, consistingessentially of, or consisting of an AAV capsid coding sequence that isat least 70% identical to: (a) the nucleotide sequence of any one of SEQID NOS:1-43; or (b) a nucleotide sequence encoding any one of SEQ IDNOS:44-86; and viruses comprising the chimeric AAV capsids. In someembodiments, the AAV capsid coding sequence is at least 70%, 71%, 72%,73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%identical to the nucleotide sequence of (a) or (b). In anotherembodiment, the AAV capsid coding sequence comprises, consistessentially of, or consist of the nucleotide sequence of (a) or (b).

In certain embodiments, the invention relates to a nucleic acid encodingan AAV capsid, the nucleic acid comprising, consisting essentially of,or consisting of the VP1, VP2, or VP1/VP2 encoding portion of an AAVcapsid coding sequence that is at least 70% identical to: (a) thenucleotide sequence of any one of SEQ ID NOS:1-43; or (b) a nucleotidesequence encoding any one of SEQ ID NOS:44-86; operably linked to theVP3 encoding portion of a different AAV capsid coding sequence; andviruses comprising the chimeric AAV capsids. In some embodiments, theVP1, VP2, or VP1/VP2 encoding portion of the AAV capsid coding sequenceis at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% identical to the VP1, VP2, or VP1/VP2 encodingportion of the nucleotide sequence of (a) or (b). In another embodiment,the VP1, VP2, or VP1/VP2 encoding portion of the AAV capsid codingsequence comprises, consists essentially of, or consists of the VP1,VP2, or VP1/VP2 encoding portion of the nucleotide sequence of (a) or(b). In some embodiments, the VP3 encoding portion of a different AAVcapsid coding sequence is a wild-type capsid sequence (e.g., AAV8 orAAV9) or a chimeric sequence that is different from any of the capsidsequences of the present invention.

Another aspect of the invention relates to chimeric AAV capsidstructures capable of providing CNS gene transfer in a subject having aCNS disorder, e.g., a neurodevelopmental disorder, in particular Rettsyndrome, e.g., a disorder caused by a mutation in the gene (MECP2)encoding methyl cytosine binding protein 2. In certain embodiments, theinvention relates to a nucleic acid encoding an AAV capsid, the nucleicacid comprising, consisting essentially of, or consisting of an AAVcapsid coding sequence that is at least 70% identical to: (a) thenucleotide sequence of any one of SEQ ID NOS: 87-107; or (b) anucleotide sequence encoding any one of SEQ ID NOS: 108-128; and virusescomprising the chimeric AAV capsids. In some embodiments, the AAV capsidcoding sequence is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%,79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the nucleotide sequenceof (a) or (b). In another embodiment, the AAV capsid coding sequencecomprises, consist essentially of, or consist of the nucleotide sequenceof (a) or (b).

In certain embodiments, the invention relates to a nucleic acid encodingan AAV capsid, the nucleic acid comprising, consisting essentially of,or consisting of the VP1, VP2, or VP1/VP2 encoding portion of an AAVcapsid coding sequence that is at least 70% identical to: (a) thenucleotide sequence of any one of SEQ ID NOS: 87-107; or (b) anucleotide sequence encoding any one of SEQ ID NOS: 108-128; operablylinked to the VP3 encoding portion of a different AAV capsid codingsequence; and viruses comprising the chimeric AAV capsids. In someembodiments, the VP1, VP2, or VP1/VP2 encoding portion of the AAV capsidcoding sequence is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%,79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the VP1, VP2, orVP1/VP2 encoding portion of the nucleotide sequence of (a) or (b). Inanother embodiment, the VP1, VP2, or VP1/VP2 portion of the AAV capsidcoding sequence comprises, consists essentially of, or consists of theVP1, VP2, or VP1/VP2 encoding portion of the nucleotide sequence of (a)or (b). In some embodiments, the VP3 encoding portion of a different AAVcapsid coding sequence is a wild-type capsid sequence (e.g., AAV8 orAAV9) or a chimeric sequence that is different from any of the capsidsequences of the present invention.

SEQ ID NOS:44-86 and 108-128 show the VP1 capsid protein sequence. Thedesignation of all amino acid positions in the description of theinvention and the appended claims is with respect to VP1 numbering.Those skilled in the art will understand that the AAV capsid generallycontains the smaller VP2 and VP3 capsid proteins as well. Due to theoverlap of the coding sequences for the AAV capsid proteins, the nucleicacid coding sequences and amino acid sequences of the VP2 and VP3 capsidproteins will be apparent from the VP1 sequences shown in the disclosedsequences. In particular, VP2 starts at nucleotide 412 (acg) of SEQ IDNO:1 and threonine 138 of SEQ ID NO:44. VP3 starts at nucleotide 607(atg) of SEQ ID NO:1 and methionine 203 of SEQ ID NO:44. In certainembodiments, isolated VP2 and VP3 capsid proteins comprising thesequence from SEQ ID NO:44 and isolated nucleic acids encoding the VP2or VP3 proteins, or both, are contemplated.

The invention also provides chimeric AAV capsid proteins and chimericcapsids, wherein the capsid protein comprises, consists essentially of,or consists of an amino acid sequence as shown in SEQ ID NOS:44-86 and108-128, wherein 1, 2 or fewer, 3 or fewer, 4 or fewer, 5 or fewer, 6 orfewer, 7 or fewer, 8 or fewer, 9 or fewer, 10 or fewer, 12 or fewer, 15or fewer, 20 or fewer, 25 or fewer, 30 or fewer, 40 or fewer, or 50 orfewer of the amino acids within the capsid protein coding sequence ofSEQ ID NOS:44-86 and 108-128 is substituted by another amino acid(naturally occurring, modified and/or synthetic), optionally aconservative amino acid substitution, and/or are deleted and/or thereare insertions (including N-terminal and C-terminal extensions) of 1, 2or fewer, 3 or fewer, 4 or fewer, 5 or fewer, 6 or fewer, 7 or fewer, 8or fewer, 9 or fewer, 10 or fewer, 12 or fewer, 15 or fewer, 20 orfewer, 25 or fewer, 30 or fewer, 40 or fewer, or 50 or fewer amino acidsor any combination of substitutions, deletions and/or insertions,wherein the substitutions, deletions and/or insertions do not undulyimpair the structure and/or function of a virion (e.g., an AAV virion)comprising the variant capsid protein or capsid. For example, inrepresentative embodiments of the invention, an AAV virion comprisingthe chimeric capsid protein substantially retains at least one propertyof a chimeric virion comprising a chimeric capsid protein as shown inSEQ ID NOS:44-86 and 108-128. For example, the virion comprising thechimeric capsid protein can substantially retain the CNS tropism profileof a virion comprising the chimeric AAV capsid protein as shown in SEQID NOS:44-86 and 108-128. Methods of evaluating biological propertiessuch as virus transduction are well-known in the art (see, e.g., theExamples).

Conservative amino acid substitutions are known in the art. Inparticular embodiments, a conservative amino acid substitution includessubstitutions within one or more of the following groups: glycine,alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid;asparagine, glutamine; serine, threonine; lysine, arginine; and/orphenylalanine, tyrosine.

It will be apparent to those skilled in the art that the amino acidsequences of the chimeric AAV capsid protein of SEQ ID NOS:44-86 and108-128 can further be modified to incorporate other modifications asknown in the art to impart desired properties. As nonlimitingpossibilities, the capsid protein can be modified to incorporatetargeting sequences (e.g., RGD) or sequences that facilitatepurification and/or detection. For example, the capsid protein can befused to all or a portion of glutathione-S-transferase, maltose-bindingprotein, a heparin/heparan sulfate binding domain, poly-His, a ligand,and/or a reporter protein (e.g., Green Fluorescent Protein,β-glucuronidase, β-galactosidase, luciferase, etc.), an immunoglobulinFc fragment, a single-chain antibody, hemagglutinin, c-myc, FLAGepitope, and the like to form a fusion protein. Methods of insertingtargeting peptides into the AAV capsid are known in the art (see, e.g.,international patent publication WO 00/28004; Nicklin et al., (2001)Mol.Ther. 474-181; White et al., (2004) Circulation 109:513-319; Muller etal., (2003) Nature Biotech. 21:1040-1046.

The viruses of the invention can further comprise a duplexed viralgenome as described in international patent publication WO 01/92551 andU.S. Pat. No. 7,465,583.

The invention also provides AAV capsids comprising the chimeric AAVcapsid proteins of the invention and virus particles (i.e., virions)comprising the same, wherein the virus particle packages (i.e.,encapsidates) a vector genome, optionally an AAV vector genome. Inparticular embodiments, the invention provides an AAV particlecomprising an AAV capsid comprising an AAV capsid protein of theinvention, wherein the AAV capsid packages an AAV vector genome. Theinvention also provides an AAV particle comprising an AAV capsid or AAVcapsid protein encoded by the chimeric nucleic acid capsid codingsequences of the invention.

In particular embodiments, the virion is a recombinant vector comprisinga heterologous nucleic acid of interest, e.g., for delivery to a cell.Thus, the present invention is useful for the delivery of nucleic acidsto cells in vitro, ex vivo, and in vivo. In representative embodiments,the recombinant vector of the invention can be advantageously employedto deliver or transfer nucleic acids to animal (e.g., mammalian) cells.

Any heterologous nucleotide sequence(s) may be delivered by a virusvector of the present invention. Nucleic acids of interest includenucleic acids encoding polypeptides, optionally therapeutic (e.g., formedical or veterinary uses) and/or immunogenic (e.g., for vaccines)polypeptides.

In some embodiments, the polypeptide is one that stimulates growthand/or differentiation of CNS cells, e.g., neurons, glial cells,oligodendrocytes, astrocytes, microglia, and/or ependymal cells.Examples include, without limitation, insulin-like growth factor-1,glial-derived neurotrophic factor, neurotrophin-3, neurotrophin-4,artemin, neurterin, persephin, brain-derived neurotrophic factor, nervegrowth factor, ciliary neurotrophic factor, transforming growth factoralpha, platelet-derived growth factor, leukemia inhibitory factor,prolactin, monocarboxylate transporter 1, or nuclear factor 1A.

Therapeutic polypeptides include, but are not limited to, cysticfibrosis transmembrane regulator protein (CFTR), dystrophin (includingthe protein product of dystrophin mini-genes or micro-genes, see, e.g.,Vincent et al., (1993) Nature Genetics 5:130; U.S. Patent PublicationNo. 2003017131; Wang et al., (2000) Proc. Natl. Acad. Sci. USA97:13714-9 [mini-dystrophin]; Harper et al., (2002) Nature Med. 8:253-61[micro-dystrophin]); mini-agrin, a laminin-α2, a sarcoglycan (α, β, γ orδ), Fukutin-related protein, myostatin pro-peptide, follistatin,dominant negative myostatin, an angiogenic factor (e.g., VEGF,angiopoietin-1 or 2), an anti-apoptotic factor (e.g., heme-oxygenase-1,TGF-β, inhibitors of pro-apoptotic signals such as caspases, proteases,kinases, death receptors [e.g., CD-095], modulators of cytochrome Crelease, inhibitors of mitochondrial pore opening and swelling); activintype II soluble receptor, anti-inflammatory polypeptides such as theIkappa B dominant mutant, sarcospan, utrophin, mini-utrophin, antibodiesor antibody fragments against myostatin or myostatin propeptide, cellcycle modulators, Rho kinase modulators such as Cethrin, which is amodified bacterial C3 exoenzyme [available from BioAxone Therapeutics,Inc., Saint-Lauren, Quebec, Canada], BCL-xL, BCL2, XIAP, FLICEc-s,dominant-negative caspase-8, dominant negative caspase-9, SPI-6 (see,e.g., U.S. Patent Application No. 20070026076), transcriptional factorPGC-α1, Pinch gene, ILK gene and thymosin 134 gene), clotting factors(e.g., Factor VIII, Factor IX, Factor X, etc.), erythropoietin,angiostatin, endostatin, catalase, tyrosine hydroxylase, anintracellular and/or extracellular superoxide dismutase, leptin, the LDLreceptor, neprilysin, lipoprotein lipase, ornithine transcarbamylase,β-globin, α-globin, spectrin, α₁-antitrypsin, methyl cytosine bindingprotein 2, adenosine deaminase, hypoxanthine guanine phosphoribosyltransferase, β-glucocerebrosidase, sphingomyelinase, lysosomalhexosaminidase A, branched-chain keto acid dehydrogenase, RP65 protein,a cytokine (e.g., α-interferon, β-interferon, interferon-γ,interleukins-1 through -14, granulocyte-macrophage colony stimulatingfactor, lymphotoxin, and the like), peptide growth factors, neurotrophicfactors and hormones (e.g., somatotropin, insulin, insulin-like growthfactors including IGF-1 and IGF-2, GLP-1, platelet derived growthfactor, epidermal growth factor, fibroblast growth factor, nerve growthfactor, neurotrophic factor −3 and −4, brain-derived neurotrophicfactor, glial derived growth factor, transforming growth factor −α and−β, and the like), bone morphogenic proteins (including RANKL and VEGF),a lysosomal protein, a glutamate receptor, a lymphokine, soluble CD4, anFc receptor, a T cell receptor, ApoE, ApoC, inhibitor 1 of proteinphosphatase inhibitor 1 (I-1), phospholamban, serca2a, lysosomal acidα-glucosidase, α-galactosidase A, Barkct, β2-adrenergic receptor,β2-adrenergic receptor kinase (BARK), phosphoinositide-3 kinase (PI3kinase), calsarcin, a receptor (e.g., the tumor necrosis growth factor-αsoluble receptor), an anti-inflammatory factor such as IRAP, Pim-1,PGC-1α, SOD-1, SOD-2, ECF-SOD, kallikrein, thymosin-J34,hypoxia-inducible transcription factor [HIF], an angiogenic factor,S100A1, parvalbumin, adenylyl cyclase type 6, a molecule that effectsG-protein coupled receptor kinase type 2 knockdown such as a truncatedconstitutively active bARKct; phospholamban inhibitory ordominant-negative molecules such as phospholamban S16E, a monoclonalantibody (including single chain monoclonal antibodies) or a suicidegene product (e.g., thymidine kinase, cytosine deaminase, diphtheriatoxin, and tumor necrosis factors such as TNF-α), and any otherpolypeptide that has a therapeutic effect in a subject in need thereof.

Heterologous nucleotide sequences encoding polypeptides include thoseencoding reporter polypeptides (e.g., an enzyme). Reporter polypeptidesare known in the art and include, but are not limited to, a fluorescentprotein (e.g., EGFP, GFP, RFP, BFP, YFP, or dsRED2), an enzyme thatproduces a detectable product, such as luciferase (e.g., from Gaussia,Renilla, or Photinus), β-galactosidase, β-glucuronidase, alkalinephosphatase, and chloramphenicol acetyltransferase gene, or proteinsthat can be directly detected. Virtually any protein can be directlydetected by using, for example, specific antibodies to the protein.Additional markers (and associated antibiotics) that are suitable foreither positive or negative selection of eukaryotic cells are disclosedin Sambrook and Russell (2001), Molecular Cloning, 3rd Ed., Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., and Ausubel et al.(1992), Current Protocols in Molecular Biology, John Wiley & Sons,including periodic updates.

Alternatively, the heterologous nucleic acid may encode a functionalRNA, e.g., an antisense oligonucleotide, a ribozyme (e.g., as describedin U.S. Pat. No. 5,877,022), RNAs that effect spliceosome-mediatedtrans-splicing (see, Puttaraju et al., (1999) Nature Biotech. 17:246;U.S. Pat. Nos. 6,013,487; 6,083,702), interfering RNAs (RNAi) includingsmall interfering RNAs (siRNA) that mediate gene silencing (see, Sharpet al., (2000) Science 287:2431), microRNA, or other non-translated“functional” RNAs, such as “guide” RNAs (Gorman et al., (1998) Proc.Nat. Acad. Sci. USA 95:4929; U.S. Pat. No. 5,869,248 to Yuan et al.),and the like. Exemplary untranslated RNAs include RNAi or antisense RNAagainst the multiple drug resistance (MDR) gene product (e.g., to treattumors and/or for administration to the heart to prevent damage bychemotherapy), RNAi or antisense RNA against myostatin (Duchenne orBecker muscular dystrophy), RNAi or antisense RNA against VEGF or atumor immunogen including but not limited to those tumor immunogensspecifically described herein (to treat tumors), RNAi or antisenseoligonucleotides targeting mutated dystrophins (Duchenne or Beckermuscular dystrophy), RNAi or antisense RNA against the hepatitis Bsurface antigen gene (to prevent and/or treat hepatitis B infection),RNAi or antisense RNA against the HIV tat and/or rev genes (to preventand/or treat HIV) and/or RNAi or antisense RNA against any otherimmunogen from a pathogen (to protect a subject from the pathogen) or adefective gene product (to prevent or treat disease). RNAi or antisenseRNA against the targets described above or any other target can also beemployed as a research reagent.

As is known in the art, anti-sense nucleic acids (e.g., DNA or RNA) andinhibitory RNA (e.g., microRNA and RNAi such as siRNA or shRNA)sequences can be used to induce “exon skipping” in patients withmuscular dystrophy arising from defects in the dystrophin gene. Thus,the heterologous nucleic acid can encode an antisense nucleic acid orinhibitory RNA that induces appropriate exon skipping. Those skilled inthe art will appreciate that the particular approach to exon skippingdepends upon the nature of the underlying defect in the dystrophin gene,and numerous such strategies are known in the art. Exemplary antisensenucleic acids and inhibitory RNA sequences target the upstream branchpoint and/or downstream donor splice site and/or internal splicingenhancer sequence of one or more of the dystrophin exons (e.g., exons 19or 23). For example, in particular embodiments, the heterologous nucleicacid encodes an antisense nucleic acid or inhibitory RNA directedagainst the upstream branch point and downstream splice donor site ofexon 19 or 23 of the dystrophin gene. Such sequences can be incorporatedinto an AAV vector delivering a modified U7 snRNA and the antisensenucleic acid or inhibitory RNA (see, e.g., Goyenvalle et al., (2004)Science 306:1796-1799). As another strategy, a modified U1 snRNA can beincorporated into an AAV vector along with siRNA, microRNA or antisenseRNA complementary to the upstream and downstream splice sites of adystrophin exon (e.g., exon 19 or 23) (see, e.g., Denti et al., (2006)Proc. Nat. Acad. Sci. USA 103:3758-3763). Further, antisense nucleicacids and inhibitory RNA can target the splicing enhancer sequenceswithin exons 19, 43, 45 or 53 (see, e.g., U.S. Pat. Nos. 6,653,467;6,727,355; and 6,653,466).

Ribozymes are RNA-protein complexes that cleave nucleic acids in asite-specific fashion. Ribozymes have specific catalytic domains thatpossess endonuclease activity (Kim et al., (1987) Proc. Natl. Acad. Sci.USA 84:8788; Gerlach et al., (1987) Nature 328:802; Forster and Symons,(1987) Cell 49:211). For example, a large number of ribozymes acceleratephosphoester transfer reactions with a high degree of specificity, oftencleaving only one of several phosphoesters in an oligonucleotidesubstrate (Michel and Westhof, (1990) J Mol. Biol. 216:585;Reinhold-Hurek and Shub, (1992) Nature 357:173). This specificity hasbeen attributed to the requirement that the substrate bind via specificbase-pairing interactions to the internal guide sequence (“IGS”) of theribozyme prior to chemical reaction.

Ribozyme catalysis has primarily been observed as part ofsequence-specific cleavage/ligation reactions involving nucleic acids(Joyce, (1989) Nature 338:217). For example, U.S. Pat. No. 5,354,855reports that certain ribozymes can act as endonucleases with a sequencespecificity greater than that of known ribonucleases and approachingthat of the DNA restriction enzymes. Thus, sequence-specificribozyme-mediated inhibition of nucleic acid expression may beparticularly suited to therapeutic applications (Scanlon et al., (1991)Proc. Natl. Acad. Sci. USA 88:10591; Sarver et al., (1990) Science247:1222; Sioud et al., (1992) J Mol. Biol. 223:831).

MicroRNAs (mir) are natural cellular RNA molecules that can regulate theexpression of multiple genes by controlling the stability of the mRNA.Over-expression or diminution of a particular microRNA can be used totreat a dysfunction and has been shown to be effective in a number ofdisease states and animal models of disease (see, e.g., Couzin, (2008)Science 319:1782-4). The chimeric AAV can be used to deliver microRNAinto cells, tissues and subjects for the treatment of genetic andacquired diseases, or to enhance functionality and promote growth ofcertain tissues. For example, mir-1, mir-133, mir-206 and/or mir-208 canbe used to treat cardiac and skeletal muscle disease (see, e.g., Chen etal., (2006) Genet. 38:228-33; van Rooij et al., (2008) Trends Genet.24:159-66). MicroRNA can also be used to modulate the immune systemafter gene delivery (Brown et al., (2007) Blood 110:4144-52).

The term “antisense oligonucleotide” (including “antisense RNA”) as usedherein, refers to a nucleic acid that is complementary to andspecifically hybridizes to a specified DNA or RNA sequence. Antisenseoligonucleotides and nucleic acids that encode the same can be made inaccordance with conventional techniques. See, e.g., U.S. Pat. No.5,023,243 to Tullis; U.S. Pat. No. 5,149,797 to Pederson et al.

Those skilled in the art will appreciate that it is not necessary thatthe antisense oligonucleotide be fully complementary to the targetsequence as long as the degree of sequence similarity is sufficient forthe antisense nucleotide sequence to specifically hybridize to itstarget (as defined above) and reduce production of the protein product(e.g., by at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% ormore).

To determine the specificity of hybridization, hybridization of sucholigonucleotides to target sequences can be carried out under conditionsof reduced stringency, medium stringency or even stringent conditions.Suitable conditions for achieving reduced, medium and stringenthybridization conditions are as described herein.

Alternatively stated, in particular embodiments, antisenseoligonucleotides of the invention have at least about 60%, 70%, 80%,90%, 95%, 97%, 98% or higher sequence identity with the complement ofthe target sequence and reduce production of the protein product (asdefined above). In some embodiments, the antisense sequence contains 1,2, 3, 4, 5, 6, 7, 8, 9 or 10 mismatches as compared with the targetsequence.

Methods of determining percent identity of nucleic acid sequences aredescribed in more detail elsewhere herein.

The length of the antisense oligonucleotide is not critical as long asit specifically hybridizes to the intended target and reduces productionof the protein product (as defined above) and can be determined inaccordance with routine procedures. In general, the antisenseoligonucleotide is at least about eight, ten or twelve or fifteennucleotides in length and/or less than about 20, 30, 40, 50, 60, 70, 80,100 or 150 nucleotides in length.

RNA interference (RNAi) is another useful approach for reducingproduction of a protein product (e.g., shRNA or siRNA). RNAi is amechanism of post-transcriptional gene silencing in whichdouble-stranded RNA (dsRNA) corresponding to a target sequence ofinterest is introduced into a cell or an organism, resulting indegradation of the corresponding mRNA. The mechanism by which RNAiachieves gene silencing has been reviewed in Sharp et al., (2001) GenesDev 15: 485-490; and Hammond et al., (2001) Nature Rev. Gen. 2:110-119).The RNAi effect persists for multiple cell divisions before geneexpression is regained. RNAi is therefore a powerful method for makingtargeted knockouts or “knockdowns” at the RNA level. RNAi has provensuccessful in human cells, including human embryonic kidney and HeLacells (see, e.g., Elbashir et al., Nature (2001) 411:494-8).

Initial attempts to use RNAi in mammalian cells resulted in antiviraldefense mechanisms involving PKR in response to the dsRNA molecules(see, e.g., Gil et al., (2000) Apoptosis 5:107). It has since beendemonstrated that short synthetic dsRNA of about 21 nucleotides, knownas “short interfering RNAs” (siRNA) can mediate silencing in mammaliancells without triggering the antiviral response (see, e.g., Elbashir etal., Nature (2001) 411:494-8; Caplen et al., (2001) Proc. Nat. Acad.Sci. USA 98:9742).

The RNAi molecule (including an siRNA molecule) can be a short hairpinRNA (shRNA; see Paddison et al., (2002), Proc. Nat. Acad. Sci. USA99:1443-1448), which is believed to be processed in the cell by theaction of the RNase III like enzyme Dicer into 20-25 mer siRNAmolecules. The shRNAs generally have a stem-loop structure in which twoinverted repeat sequences are separated by a short spacer sequence thatloops out. There have been reports of shRNAs with loops ranging from 3to 23 nucleotides in length. The loop sequence is generally notcritical. Exemplary loop sequences include the following motifs: AUG,CCC, UUCG, CCACC, CTCGAG, AAGCUU, CCACACC and UUCAAGAGA.

The RNAi can further comprise a circular molecule comprising sense andantisense regions with two loop regions on either side to form a“dumbbell” shaped structure upon dsRNA formation between the sense andantisense regions. This molecule can be processed in vitro or in vivo torelease the dsRNA portion, e.g., a siRNA.

International patent publication WO 01/77350 describes a vector forbi-directional transcription to generate both sense and antisensetranscripts of a heterologous sequence in a eukaryotic cell. Thistechnique can be employed to produce RNAi for use according to theinvention.

Shinagawa et al., (2003) Genes Dev. 17:1340 reported a method ofexpressing long dsRNAs from a CMV promoter (a pol II promoter), whichmethod is also applicable to tissue specific pol II promoters. Likewise,the approach of Xia et al., (2002) Nature Biotech. 20:1006, avoidspoly(A) tailing and can be used in connection with tissue-specificpromoters.

Methods of generating RNAi include chemical synthesis, in vitrotranscription, digestion of long dsRNA by Dicer (in vitro or in vivo),expression in vivo from a delivery vector, and expression in vivo from aPCR-derived RNAi expression cassette (see, e.g., TechNotes 10(3) “FiveWays to Produce siRNAs,” from Ambion, Inc., Austin Tex.; available atwww.ambion.com).

Guidelines for designing siRNA molecules are available (see e.g.,literature from Ambion, Inc., Austin Tex.; available at www.ambion.com).In particular embodiments, the siRNA sequence has about 30-50% G/Ccontent. Further, long stretches of greater than four T or A residuesare generally avoided if RNA polymerase III is used to transcribe theRNA. Online siRNA target finders are available, e.g., from Ambion, Inc.(www.ambion.com), through the Whitehead Institute of Biomedical Research(www.jura.wi.mit.edu) or from Dharmacon Research, Inc.(www.dharmacon.com).

The antisense region of the RNAi molecule can be completelycomplementary to the target sequence, but need not be as long as itspecifically hybridizes to the target sequence (as defined above) andreduces production of the protein product (e.g., by at least about 30%,40%, 50%, 60%, 70%, 80%, 90%, 95% or more). In some embodiments,hybridization of such oligonucleotides to target sequences can becarried out under conditions of reduced stringency, medium stringency oreven stringent conditions, as defined above.

In other embodiments, the antisense region of the RNAi has at leastabout 60%, 70%, 80%, 90%, 95%, 97%, 98% or higher sequence identity withthe complement of the target sequence and reduces production of theprotein product (e.g., by at least about 30%, 40%, 50%, 60%, 70%, 80%,90%, 95% or more). In some embodiments, the antisense region contains 1,2, 3, 4, 5, 6, 7, 8, 9 or 10 mismatches as compared with the targetsequence. Mismatches are generally tolerated better at the ends of thedsRNA than in the center portion.

In particular embodiments, the RNAi is formed by intermolecularcomplexing between two separate sense and antisense molecules. The RNAicomprises a ds region formed by the intermolecular basepairing betweenthe two separate strands. In other embodiments, the RNAi comprises a dsregion formed by intramolecular basepairing within a single nucleic acidmolecule comprising both sense and antisense regions, typically as aninverted repeat (e.g., a shRNA or other stem loop structure, or acircular RNAi molecule). The RNAi can further comprise a spacer regionbetween the sense and antisense regions.

Generally, RNAi molecules are highly selective. If desired, thoseskilled in the art can readily eliminate candidate RNAi that are likelyto interfere with expression of nucleic acids other than the target bysearching relevant databases to identify RNAi sequences that do not havesubstantial sequence homology with other known sequences, for example,using BLAST (available at www.ncbi.nlm.nih.gov/BLAST).

Kits for the production of RNAi are commercially available, e.g., fromNew England Biolabs, Inc. and Ambion, Inc.

The recombinant virus vector may also comprise a heterologous nucleotidesequence that shares homology with and recombines with a locus on thehost chromosome. This approach may be utilized to correct a geneticdefect in the host cell.

The present invention also provides recombinant virus vectors thatexpress an immunogenic polypeptide, e.g., for vaccination. Theheterologous nucleic acid may encode any immunogen of interest known inthe art including, but are not limited to, immunogens from humanimmunodeficiency virus, influenza virus, gag proteins, tumor antigens,cancer antigens, bacterial antigens, viral antigens, and the like.Alternatively, the immunogen can be presented in the virus capsid (e.g.,incorporated therein) or tethered to the virus capsid (e.g., by covalentmodification).

The use of parvoviruses as vaccines is known in the art (see, e.g.,Miyamura et al., (1994) Proc. Nat. Acad. Sci. USA 91:8507; U.S. Pat. No.5,916,563 to Young et al., U.S. Pat. No. 5,905,040 to Mazzara et al.,U.S. Pat. Nos. 5,882,652, 5,863,541 to Samulski et al.; the disclosuresof which are incorporated herein in their entireties by reference). Theantigen may be presented in the virus capsid. Alternatively, the antigenmay be expressed from a heterologous nucleic acid introduced into arecombinant vector genome.

An immunogenic polypeptide, or immunogen, may be any polypeptidesuitable for protecting the subject against a disease, including but notlimited to microbial, bacterial, protozoal, parasitic, fungal and viraldiseases. For example, the immunogen may be an orthomyxovirus immunogen(e.g., an influenza virus immunogen, such as the influenza virushemagglutinin (HA) surface protein or the influenza virus nucleoproteingene, or an equine influenza virus immunogen), or a lentivirus immunogen(e.g., an equine infectious anemia virus immunogen, a SimianImmunodeficiency Virus (SIV) immunogen, or a Human ImmunodeficiencyVirus (HIV) immunogen, such as the HIV or SIV envelope GP160 protein,the HIV or SIV matrix/capsid proteins, and the HIV or SIV gag, pol andenv genes products). The immunogen may also be an arenavirus immunogen(e.g., Lassa fever virus immunogen, such as the Lassa fever virusnucleocapsid protein gene and the Lassa fever envelope glycoproteingene), a poxvirus immunogen (e.g., vaccinia, such as the vaccinia L1 orL8 genes), a flavivirus immunogen (e.g., a yellow fever virus immunogenor a Japanese encephalitis virus immunogen), a Filovirus immunogen(e.g., an Ebola virus immunogen, or a Marburg virus immunogen, such asNP and GP genes), a bunyavirus immunogen (e.g., RVFV, CCHF, and SFSviruses), or a coronavirus immunogen (e.g., an infectious humancoronavirus immunogen, such as the human coronavirus envelopeglycoprotein gene, or a porcine transmissible gastroenteritis virusimmunogen, or an avian infectious bronchitis virus immunogen, or asevere acute respiratory syndrome (SARS) immunogen such as a S [S1 orS2], M, E, or N protein or an immunogenic fragment thereof). Theimmunogen may further be a polio immunogen, herpes immunogen (e.g., CMV,EBV, HSV immunogens) mumps immunogen, measles immunogen, rubellaimmunogen, diphtheria toxin or other diphtheria immunogen, pertussisantigen, hepatitis (e.g., hepatitis A, hepatitis B or hepatitis C)immunogen, or any other vaccine immunogen known in the art.

Alternatively, the immunogen may be any tumor or cancer cell antigen.Optionally, the tumor or cancer antigen is expressed on the surface ofthe cancer cell. Exemplary cancer and tumor cell antigens are describedin S. A. Rosenberg, (1999) Immunity 10:281). Illustrative cancer andtumor antigens include, but are not limited to: BRCA1 gene product,BRCA2 gene product, gp100, tyrosinase, GAGE-1/2, BAGE, RAGE, NY-ESO-1,CDK-4, β-catenin, MUM-1, Caspase-8, KIAA0205, HPVE, SART-1, PRAIVIE,p15, melanoma tumor antigens (Kawakami et al., (1994) Proc. Natl. Acad.Sci. USA 91:3515; Kawakami et al., (1994) J. Exp. Med., 180:347;Kawakami et al., (1994) Cancer Res. 54:3124) including MART-1 (Coulie etal., (1991) J Exp. Med. 180:35), gp100 (Wick et al., (1988) J. Cutan.Pathol. 4:201) and MAGE antigen (MAGE-1, MAGE-2 and MAGE-3) (Van derBruggen et al., (1991) Science, 254:1643), CEA, TRP-1; TRP-2; P-15 andtyrosinase (Brichard et al., (1993) J. Exp. Med. 178:489); HER-2/neugene product (U.S. Pat. No. 4,968,603); CA 125; HE4; LK26; FB5(endosialin); TAG 72; AFP; CA19-9; NSE; DU-PAN-2; CA50; Span-1; CA72-4;HCG; STN (sialyl Tn antigen); c-erbB-2 proteins; PSA; L-CanAg; estrogenreceptor; milk fat globulin; p53 tumor suppressor protein (Levine,(1993) Ann. Rev. Biochem. 62:623); mucin antigens (international patentpublication WO 90/05142); telomerases; nuclear matrix proteins;prostatic acid phosphatase; papilloma virus antigens; and antigensassociated with the following cancers: melanomas, adenocarcinoma,thymoma, sarcoma, lung cancer, liver cancer, colorectal cancer,non-Hodgkin's lymphoma, Hodgkin's lymphoma, leukemias, uterine cancer,breast cancer, prostate cancer, ovarian cancer, cervical cancer, bladdercancer, kidney cancer, pancreatic cancer, brain cancer, kidney cancer,stomach cancer, esophageal cancer, head and neck cancer and others (see,e.g., Rosenberg, (1996) Annu. Rev. Med. 47:481-91).

Alternatively, the heterologous nucleotide sequence may encode anypolypeptide that is desirably produced in a cell in vitro, ex vivo, orin vivo. For example, the virus vectors may be introduced into culturedcells and the expressed protein product isolated therefrom.

It will be understood by those skilled in the art that the heterologousnucleic acid(s) of interest may be operably associated with appropriatecontrol sequences. For example, the heterologous nucleic acid may beoperably associated with expression control elements, such astranscription/translation control signals, origins of replication,polyadenylation signals, internal ribosome entry sites (IRES),promoters, enhancers, and the like.

Those skilled in the art will further appreciate that a variety ofpromoter/enhancer elements may be used depending on the level andtissue-specific expression desired. The promoter/enhancer may beconstitutive or inducible, depending on the pattern of expressiondesired. The promoter/enhancer may be native or foreign and can be anatural or a synthetic sequence. By foreign, it is intended that thetranscriptional initiation region is not found in the wild-type hostinto which the transcriptional initiation region is introduced.

Promoter/enhancer elements can be native to the target cell or subjectto be treated and/or native to the heterologous nucleic acid sequence.The promoter/enhancer element is generally chosen so that it willfunction in the target cell(s) of interest. In representativeembodiments, the promoter/enhancer element is a mammalianpromoter/enhancer element. The promoter/enhance element may beconstitutive or inducible.

Inducible expression control elements are generally used in thoseapplications in which it is desirable to provide regulation overexpression of the heterologous nucleic acid sequence(s). Induciblepromoters/enhancer elements for gene delivery can be tissue-specific ortissue-preferred promoter/enhancer elements, and include muscle specificor preferred (including cardiac, skeletal and/or smooth muscle), neuraltissue specific or preferred (including brain-specific), eye (includingretina-specific and cornea-specific), liver specific or preferred, bonemarrow specific or preferred, pancreatic specific or preferred, spleenspecific or preferred, and lung specific or preferred promoter/enhancerelements. In one embodiment, a CNS cell-specific or CNS cell-preferredpromoter is used. Examples of neuron-specific or preferred promotersinclude, without limitation, neuronal-specific enolase, synapsin, andMeCP2. Examples of astrocyte-specific or preferred promoters include,without limitation, glial fibrillary acidic protein and S1000. Examplesof ependymal cell-specific or preferred promoters include, withoutlimitation, wdr16, Foxj 1, and LRP2. Examples of microglia-specific orpreferred promoters include, without limitation, F4/80, CX3CR1, andCD11b. Examples of oligodendrocyte-specific or preferred promotersinclude, without limitation, myelin basic protein, cyclic nucleotidephosphodiesterase, proteolipid protein, Gtx, and Sox10. Use of a CNScell-specific or preferred promoter can increase the specificityachieved by the chimeric AAV vector by further limiting expression ofthe heterologous nucleic acid to the CNS. Other induciblepromoter/enhancer elements include hormone-inducible and metal-inducibleelements. Exemplary inducible promoters/enhancer elements include, butare not limited to, a Tet on/off element, a RU486-inducible promoter, anecdysone-inducible promoter, a rapamycin-inducible promoter, and ametallothionein promoter.

In embodiments wherein the heterologous nucleic acid sequence(s) istranscribed and then translated in the target cells, specific initiationsignals are generally employed for efficient translation of insertedprotein coding sequences. These exogenous translational controlsequences, which may include the ATG initiation codon and adjacentsequences, can be of a variety of origins, both natural and synthetic.

The invention also provides chimeric AAV particles comprising an AAVcapsid and an AAV genome, wherein the AAV genome “corresponds to” (i.e.,encodes) the AAV capsid. Also provided are collections or libraries ofsuch chimeric AAV particles, wherein the collection or library comprises2 or more, 10 or more, 50 or more, 100 or more, 1000 or more, 10⁴ ormore, 10⁵ or more, or 10⁶ or more distinct sequences.

The present invention further encompasses “empty” capsid particles(i.e., in the absence of a vector genome) comprising, consisting of, orconsisting essentially of the chimeric AAV capsid proteins of theinvention. The chimeric AAV capsids of the invention can be used as“capsid vehicles,” as has been described in U.S. Pat. No. 5,863,541.Molecules that can be covalently linked, bound to or packaged by thevirus capsids and transferred into a cell include DNA, RNA, a lipid, acarbohydrate, a polypeptide, a small organic molecule, or combinationsof the same. Further, molecules can be associated with (e.g., “tetheredto”) the outside of the virus capsid for transfer of the molecules intohost target cells. In one embodiment of the invention the molecule iscovalently linked (i.e., conjugated or chemically coupled) to the capsidproteins. Methods of covalently linking molecules are known by thoseskilled in the art.

The virus capsids of the invention also find use in raising antibodiesagainst the novel capsid structures. As a further alternative, anexogenous amino acid sequence may be inserted into the virus capsid forantigen presentation to a cell, e.g., for administration to a subject toproduce an immune response to the exogenous amino acid sequence.

The invention also provides nucleic acids (e.g., isolated nucleic acids)encoding the chimeric virus capsids and chimeric capsid proteins of theinvention. Further provided are vectors comprising the nucleic acids,and cells (in vivo or in culture) comprising the nucleic acids and/orvectors of the invention. Such nucleic acids, vectors and cells can beused, for example, as reagents (e.g., helper constructs or packagingcells) for the production of virus vectors as described herein.

In exemplary embodiments, the invention provides nucleic acid sequencesencoding the AAV capsid of SEQ ID NOS: 44-86 or 108-128 or at least 70%identical to the nucleotide sequence of SEQ ID NOS: 1-44 or 87-107. Theinvention also provides nucleic acids encoding the AAV capsid variants,capsid protein variants and fusion proteins as described above. Inparticular embodiments, the nucleic acid hybridizes to the complement ofthe nucleic acid sequences specifically disclosed herein under standardconditions as known by those skilled in the art and encodes a variantcapsid and/or capsid protein. Optionally, the variant capsid or capsidprotein substantially retains at least one property of the capsid and/orcapsid or capsid protein encoded by the nucleic acid sequence of SEQ IDNOS: 1-44 or 87-107. For example, a virus particle comprising thevariant capsid or variant capsid protein can substantially retain theCNS tropism profile of a virus particle comprising a capsid or capsidprotein encoded by a nucleic acid coding sequence of SEQ ID NO:1-44 or87-107.

For example, hybridization of such sequences may be carried out underconditions of reduced stringency, medium stringency or even stringentconditions. Exemplary conditions for reduced, medium and stringenthybridization are as follows: (e.g., conditions represented by a washstringency of 35-40% Formamide with 5×Denhardt's solution, 0.5% SDS and1×SSPE at 37° C.; conditions represented by a wash stringency of 40-45%Formamide with 5×Denhardt's solution, 0.5% SDS, and 1×SSPE at 42° C.;and conditions represented by a wash stringency of 50% Formamide with5×Denhardt's solution, 0.5% SDS and 1×SSPE at 42° C., respectively).See, e.g., Sambrook et al., Molecular Cloning, A Laboratory Manual (2dEd. 1989) (Cold Spring Harbor Laboratory).

In other embodiments, nucleic acid sequences encoding a variant capsidor capsid protein of the invention have at least about 70%, 71%, 72%,73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, orhigher sequence identity with the nucleic acid sequence of SEQ IDNO:1-44 or 87-107 and optionally encode a variant capsid or capsidprotein that substantially retains at least one property of the capsidor capsid protein encoded by a nucleic acid of SEQ ID NO:1-44 or 87-107.

As is known in the art, a number of different programs can be used toidentify whether a nucleic acid or polypeptide has sequence identity toa known sequence. Percent identity as used herein means that a nucleicacid or fragment thereof shares a specified percent identity to anothernucleic acid, when optimally aligned (with appropriate nucleotideinsertions or deletions) with the other nucleic acid (or itscomplementary strand), using BLASTN. To determine percent identitybetween two different nucleic acids, the percent identity is to bedetermined using the BLASTN program “BLAST 2 sequences”. This program isavailable for public use from the National Center for BiotechnologyInformation (NCBI) over the Internet (Altschul et al., (1997) NucleicAcids Res. 25(17):3389-3402). The parameters to be used are whatevercombination of the following yields the highest calculated percentidentity (as calculated below) with the default parameters shown inparentheses: Program--blastn Matrix--0 BLOSUM62 Reward for a match--0 or1 (1) Penalty for a mismatch--0, -1, -2 or -3 (˜2) Open gap penalty--0,1, 2, 3, 4 or 5 (5) Extension gap penalty--0 or 1 (1) Gap x_dropoff--0or 50 (50) Expect--10.

Percent identity or similarity when referring to polypeptides, indicatesthat the polypeptide in question exhibits a specified percent identityor similarity when compared with another protein or a portion thereofover the common lengths as determined using BLASTP. This program is alsoavailable for public use from the National Center for BiotechnologyInformation (NCBI) over the Internet (Altschul et al., (1997) NucleicAcids Res. 25(17):3389-3402). Percent identity or similarity forpolypeptides is typically measured using sequence analysis software.See, e.g., the Sequence Analysis Software Package of the GeneticsComputer Group, University of Wisconsin Biotechnology Center, 910University Avenue, Madison, Wis. 53705. Protein analysis softwarematches similar sequences using measures of homology assigned to varioussubstitutions, deletions and other modifications. Conservativesubstitutions typically include substitutions within the followinggroups: glycine, alanine; valine, isoleucine, leucine; aspartic acid,glutamic acid; asparagine, glutamine; serine, threonine; lysine,arginine; and phenylalanine, tyrosine.

In particular embodiments, the nucleic acid can comprise, consistessentially of, or consist of a vector including but not limited to aplasmid, phage, viral vector (e.g., AAV vector, an adenovirus vector, aherpesvirus vector, or a baculovirus vector), bacterial artificialchromosome (BAC), or yeast artificial chromosome (YAC). For example, thenucleic acid can comprise, consist of, or consist essentially of an AAVvector comprising a 5′ and/or 3′ terminal repeat (e.g., 5′ and/or 3′ AAVterminal repeat).

In some embodiments, the nucleic acid encoding the chimeric AAV capsidprotein further comprises an AAV rep coding sequence. For example, thenucleic acid can be a helper construct for producing viral stocks.

The invention also provides packaging cells stably comprising a nucleicacid of the invention. For example, the nucleic acid can be stablyincorporated into the genome of the cell or can be stably maintained inan episomal form (e.g., an “EBV based nuclear episome”).

The nucleic acid can be incorporated into a delivery vector, such as aviral delivery vector. To illustrate, the nucleic acid of the inventioncan be packaged in an AAV particle, an adenovirus particle, aherpesvirus particle, a baculovirus particle, or any other suitablevirus particle.

Moreover, the nucleic acid can be operably associated with a promoterelement. Promoter elements are described in more detail herein.

The present invention further provides methods of producing the virusvectors of the invention. In a representative embodiment, the presentinvention provides a method of producing a recombinant virus vector, themethod comprising providing to a cell in vitro, (a) a templatecomprising (i) a heterologous nucleic acid, and (ii) packaging signalsequences sufficient for the encapsidation of the AAV template intovirus particles (e.g., one or more (e.g., two) terminal repeats, such asAAV terminal repeats), and (b) AAV sequences sufficient for replicationand encapsidation of the template into viral particles (e.g., the AAVrep and AAV cap sequences encoding an AAV capsid of the invention). Thetemplate and AAV replication and capsid sequences are provided underconditions such that recombinant virus particles comprising the templatepackaged within the capsid are produced in the cell. The method canfurther comprise the step of collecting the virus particles from thecell. Virus particles may be collected from the medium and/or by lysingthe cells.

In one illustrative embodiment, the invention provides a method ofproducing a rAAV particle comprising an AAV capsid, the methodcomprising: providing a cell in vitro with a nucleic acid encoding achimeric AAV capsid of the invention, an AAV rep coding sequence, an AAVvector genome comprising a heterologous nucleic acid, and helperfunctions for generating a productive AAV infection; and allowingassembly of the AAV particles comprising the AAV capsid andencapsidating the AAV vector genome.

The cell is typically a cell that is permissive for AAV viralreplication. Any suitable cell known in the art may be employed, such asmammalian cells. Also suitable are trans-complementing packaging celllines that provide functions deleted from a replication-defective helpervirus, e.g., 293 cells or other Ela trans-complementing cells.

The AAV replication and capsid sequences may be provided by any methodknown in the art. Current protocols typically express the AAV rep/capgenes on a single plasmid. The AAV replication and packaging sequencesneed not be provided together, although it may be convenient to do so.The AAV rep and/or cap sequences may be provided by any viral ornon-viral vector. For example, the rep/cap sequences may be provided bya hybrid adenovirus or herpesvirus vector (e.g., inserted into the Elaor E3 regions of a deleted adenovirus vector). EBV vectors may also beemployed to express the AAV cap and rep genes. One advantage of thismethod is that EBV vectors are episomal, yet will maintain a high copynumber throughout successive cell divisions (i.e., are stably integratedinto the cell as extra-chromosomal elements, designated as an EBV basednuclear episome.

As a further alternative, the rep/cap sequences may be stably carried(episomal or integrated) within a cell.

Typically, the AAV rep/cap sequences will not be flanked by the AAVpackaging sequences (e.g., AAV ITRs), to prevent rescue and/or packagingof these sequences.

The template (e.g., an rAAV vector genome) can be provided to the cellusing any method known in the art. For example, the template may besupplied by a non-viral (e.g., plasmid) or viral vector. In particularembodiments, the template is supplied by a herpesvirus or adenovirusvector (e.g., inserted into the Ela or E3 regions of a deletedadenovirus). As another illustration, Palombo et al., (1998) J. Virol.72:5025, describe a baculovirus vector carrying a reporter gene flankedby the AAV ITRs. EBV vectors may also be employed to deliver thetemplate, as described above with respect to the rep/cap genes.

In another representative embodiment, the template is provided by areplicating rAAV virus. In still other embodiments, an AAV provirus isstably integrated into the chromosome of the cell.

To obtain maximal virus titers, helper virus functions (e.g., adenovirusor herpesvirus) essential for a productive AAV infection are generallyprovided to the cell. Helper virus sequences necessary for AAVreplication are known in the art. Typically, these sequences areprovided by a helper adenovirus or herpesvirus vector. Alternatively,the adenovirus or herpesvirus sequences can be provided by anothernon-viral or viral vector, e.g., as a non-infectious adenovirusminiplasmid that carries all of the helper genes required for efficientAAV production as described by Ferrari et al., (1997) Nature Med.3:1295, and U.S. Pat. Nos. 6,040,183 and 6,093,570.

Further, the helper virus functions may be provided by a packaging cellwith the helper genes integrated in the chromosome or maintained as astable extrachromosomal element. In representative embodiments, thehelper virus sequences cannot be packaged into AAV virions, e.g., arenot flanked by AAV ITRs.

Those skilled in the art will appreciate that it may be advantageous toprovide the AAV replication and capsid sequences and the helper virussequences (e.g., adenovirus sequences) on a single helper construct.This helper construct may be a non-viral or viral construct, but isoptionally a hybrid adenovirus or hybrid herpesvirus comprising the AAVrep/cap genes.

In one particular embodiment, the AAV rep/cap sequences and theadenovirus helper sequences are supplied by a single adenovirus helpervector. This vector further contains the rAAV template. The AAV rep/capsequences and/or the rAAV template may be inserted into a deleted region(e.g., the Ela or E3 regions) of the adenovirus.

In a further embodiment, the AAV rep/cap sequences and the adenovirushelper sequences are supplied by a single adenovirus helper vector. TherAAV template is provided as a plasmid template.

In another illustrative embodiment, the AAV rep/cap sequences andadenovirus helper sequences are provided by a single adenovirus helpervector, and the rAAV template is integrated into the cell as a provirus.Alternatively, the rAAV template is provided by an EBV vector that ismaintained within the cell as an extrachromosomal element (e.g., as a“EBV based nuclear episome,” see Margolski, (1992) Curr. Top. Microbiol.Immun. 158:67).

In a further exemplary embodiment, the AAV rep/cap sequences andadenovirus helper sequences are provided by a single adenovirus helper.The rAAV template is provided as a separate replicating viral vector.For example, the rAAV template may be provided by a rAAV particle or asecond recombinant adenovirus particle.

According to the foregoing methods, the hybrid adenovirus vectortypically comprises the adenovirus 5′ and 3′ cis sequences sufficientfor adenovirus replication and packaging (i.e., the adenovirus terminalrepeats and PAC sequence). The AAV rep/cap sequences and, if present,the rAAV template are embedded in the adenovirus backbone and areflanked by the 5′ and 3′ cis sequences, so that these sequences may bepackaged into adenovirus capsids. As described above, in representativeembodiments, the adenovirus helper sequences and the AAV rep/capsequences are not flanked by the AAV packaging sequences (e.g., the AAVITRs), so that these sequences are not packaged into the AAV virions.

Herpesvirus may also be used as a helper virus in AAV packaging methods.Hybrid herpesviruses encoding the AAV rep protein(s) may advantageouslyfacilitate for more scalable AAV vector production schemes. A hybridherpes simplex virus type I (HSV-1) vector expressing the AAV-2 rep andcap genes has been described (Conway et al., (1999) Gene Therapy 6:986and WO 00/17377, the disclosures of which are incorporated herein intheir entireties).

As a further alternative, the virus vectors of the invention can beproduced in insect cells using baculovirus vectors to deliver therep/cap genes and rAAV template as described by Urabe et al., (2002)Human Gene Therapy 13:1935-43.

Other methods of producing AAV use stably transformed packaging cells(see, e.g., U.S. Pat. No. 5,658,785).

AAV vector stocks free of contaminating helper virus may be obtained byany method known in the art. For example, AAV and helper virus may bereadily differentiated based on size. AAV may also be separated awayfrom helper virus based on affinity for a heparin substrate (Zolotukhinet al., (1999) Gene Therapy 6:973). In representative embodiments,deleted replication-defective helper viruses are used so that anycontaminating helper virus is not replication competent. As a furtheralternative, an adenovirus helper lacking late gene expression may beemployed, as only adenovirus early gene expression is required tomediate packaging of AAV virus. Adenovirus mutants defective for lategene expression are known in the art (e.g., ts100K and ts149 adenovirusmutants).

The inventive packaging methods may be employed to produce high titerstocks of virus particles. In particular embodiments, the virus stockhas a titer of at least about 10⁵ transducing units (tu)/ml, at leastabout 10⁶ tu/ml, at least about 10⁷ tu/ml, at least about 10⁸ tu/ml, atleast about 10⁹ tu/ml, or at least about 10¹⁰ tu/ml.

The novel capsid protein and capsid structures find use in raisingantibodies, for example, for diagnostic or therapeutic uses or as aresearch reagent. Thus, the invention also provides antibodies againstthe novel capsid proteins and capsids of the invention.

The term “antibody” or “antibodies” as used herein refers to all typesof immunoglobulins, including IgG, IgM, IgA, IgD, and IgE. The antibodycan be monoclonal or polyclonal and can be of any species of origin,including (for example) mouse, rat, rabbit, horse, goat, sheep or human,or can be a chimeric antibody. See, e.g., Walker et al., Mol. Immunol.26, 403-11 (1989). The antibodies can be recombinant monoclonalantibodies, for example, produced according to the methods disclosed inU.S. Pat. No. 4,474,893 or 4,816,567. The antibodies can also bechemically constructed, for example, according to the method disclosedin U.S. Pat. No. 4,676,980.

Antibody fragments included within the scope of the present inventioninclude, for example, Fab, F(ab′)2, and Fc fragments, and thecorresponding fragments obtained from antibodies other than IgG. Suchfragments can be produced by known techniques. For example, F(ab′)2fragments can be produced by pepsin digestion of the antibody molecule,and Fab fragments can be generated by reducing the disulfide bridges ofthe F(ab′)2 fragments. Alternatively, Fab expression libraries can beconstructed to allow rapid and easy identification of monoclonal Fabfragments with the desired specificity (Huse et al., (1989) Science 254,1275-1281).

Polyclonal antibodies can be produced by immunizing a suitable animal(e.g., rabbit, goat, etc.) with an antigen to which a monoclonalantibody to the target binds, collecting immune serum from the animal,and separating the polyclonal antibodies from the immune serum, inaccordance with known procedures.

Monoclonal antibodies can be produced in a hybridoma cell line accordingto the technique of Kohler and Milstein, (1975) Nature 265, 495-97. Forexample, a solution containing the appropriate antigen can be injectedinto a mouse and, after a sufficient time, the mouse sacrificed andspleen cells obtained. The spleen cells are then immortalized by fusingthem with myeloma cells or with lymphoma cells, typically in thepresence of polyethylene glycol, to produce hybridoma cells. Thehybridoma cells are then grown in a suitable medium and the supernatantscreened for monoclonal antibodies having the desired specificity.Monoclonal Fab fragments can be produced in E. coli by recombinanttechniques known to those skilled in the art. See, e.g., W. Huse, (1989)Science 246, 1275-81.

Antibodies specific to a target polypeptide can also be obtained byphage display techniques known in the art.

Various immunoassays can be used for screening to identify antibodieshaving the desired specificity. Numerous protocols for competitivebinding or immunoradiometric assays using either polyclonal ormonoclonal antibodies with established specificity are well known in theart. Such immunoassays typically involve the measurement of complexformation between an antigen and its specific antibody (e.g.,antigen/antibody complex formation). A two-site, monoclonal-basedimmunoassay utilizing monoclonal antibodies reactive to twonon-interfering epitopes can be used as well as a competitive bindingassay.

Antibodies can be conjugated to a solid support (e.g., beads, plates,slides or wells formed from materials such as latex or polystyrene) inaccordance with known techniques. Antibodies can likewise be directly orindirectly conjugated to detectable groups such as radiolabels (e.g.,³⁵S, ¹²⁵I, ¹³¹I), enzyme labels (e.g., horseradish peroxidase, alkalinephosphatase), and fluorescence labels (e.g., fluorescein) in accordancewith known techniques. Determination of the formation of anantibody/antigen complex in the methods of this invention can be bydetection of, for example, precipitation, agglutination, flocculation,radioactivity, color development or change, fluorescence, luminescence,etc., as is well known in the art.

III. Methods of Using Chimeric AAV Capsids Targeted to the CNS

The present invention also relates to methods for deliveringheterologous nucleotide sequences into the CNS while minimizing deliveryto peripheral organs. The virus vectors of the invention may be employedto deliver a nucleotide sequence of interest to a CNS cell in vitro,e.g., to produce a polypeptide or nucleic acid in vitro or for ex vivogene therapy. The vectors are additionally useful in a method ofdelivering a nucleotide sequence to a subject in need thereof, e.g., toexpress a therapeutic or immunogenic polypeptide or nucleic acid. Inthis manner, the polypeptide or nucleic acid may thus be produced invivo in the subject. The subject may be in need of the polypeptide ornucleic acid because the subject has a deficiency of the polypeptide, orbecause the production of the polypeptide or nucleic acid in the subjectmay impart some therapeutic effect, as a method of treatment orotherwise, and as explained further below.

In particular embodiments, the vectors are useful to express apolypeptide or nucleic acid that provides a beneficial effect to theCNS, e.g., to promote growth and/or differentiation of neurons or glialcells. The ability to target vectors to the CNS may be particularlyuseful to treat diseases or disorders involving CNS dysfunction. Inother embodiments, the vectors are useful to express a polypeptide ornucleic acid that provides a beneficial effect to cells in the CNS(e.g., neurons and/or glial cells).

Thus, one aspect of the invention relates to a method of delivering anucleic acid of interest a CNS cell, the method comprising contactingthe CNS cell with the AAV particle of the invention.

In another aspect, the invention relates to a method of delivering anucleic acid of interest to a CNS cell in a mammalian subject, themethod comprising administering an effective amount of the AAV particleor pharmaceutical formulation of the invention to a mammalian subject.

A further aspect of the invention relates to a method of treating adisorder associated with CNS dysfunction in a subject in need thereof,the method comprising administering a therapeutically effective amountof the AAV particle of the invention to the subject.

CNS disorders include but are not limited to disorders of thinking andcognition such as schizophrenia and delirium; amnestic disorders;disorders of mood, such as affective disorders and anxiety disorders(including post-traumatic stress disorder, separation anxiety disorder,selective mutism, reactive attachment disorder, stereotypic movementdisorder, panic disorders, agoraphobia, specific phobias, social phobia,obsessive-compulsive disorder, acute stress disorder, generalizedanxiety disorder, substance-induced anxiety disorder and/or anxietydisorder not otherwise specified); disorders of social behavior;disorders of learning and memory, such as learning disorders (e.g.,dyslexia); motor skills disorders; communication disorders (e.g.,stuttering); pervasive developmental disorders (e.g., autistic disorder,Rett's disorder (Rett syndrome), childhood disintegrative disorder,Asperger's disorder, and/or pervasive developmental disorder nototherwise specified) and dementia. Accordingly, the term “centralnervous system disorder” encompasses the disorders listed above as wellas depressive disorders (including major depressive disorder, dysthmyicdisorder, depressive disorder not otherwise specified, postpartumdepression); seasonal affective disorder; mania; bipolar disorders(including bipolar I disorder, bipolar II disorder, cyclothymicdisorder, bipolar disorder not otherwise specified); attention-deficitand disruptive behavior disorders (including attention deficit disorderwith hyperactivity disorder, conduct disorder, oppositional defiantdisorder and/or disruptive behavior disorder not otherwise specified);drug addiction/substance abuse (including abuse of opiates,amphetamines, alcohol, hallucinogens, cannabis, inhalants,phencyclidine, sedatives, hypnotics, anxyolytics and/or cocaine);alcohol-induced disorders; amphetamine-induced disorders;caffeine-induced disorders; cannabis-induced disorders; cocaine-induceddisorders; hallucinogen-induced disorders; inhalant-induced disorders;nicotine-induced disorders; opioid-induced disorders;phencyclidine-induced disorders; sedative, hypnotic oranxyolytic-induced disorders; agitation; apathy; psychoses;irritability; disinhibition; schizophreniform disorder; schizoaffectivedisorder; delusional disorder; brief psychotic disorder, sharedpsychotic disorder; substance-induced psychotic disorder; psychoticdisorder not otherwise specified; unipolar disorders, mood disorders(e.g., mood disorder with psychotic features); somatoform disorders;factitious disorders; disassociative disorders; mental retardation;feeding and eating disorders of infancy or early childhood; eatingdisorders such as anorexia nervosa, bulimia nervosa and/or eatingdisorder not otherwise specified; sleeping disorders (e.g., dyssomniassuch as primary insomnia, primary hypersomnia, narcolepsy,breathing-related sleep disorder and circadian rhythm sleep disorderand/or parasomnias); impulse control disorders (e.g., kleptomania,pyromania, trichotillomania, pathological gambling and/or intermittentexplosive disorder); adjustment disorders; personality disorders (e.g.,paranoid personality disorder, schizoid personality disorder,schizotypal personality disorder, antisocial personality disorder,borderline personality disorder, histrionic personality disorder,narcissistic personality disorder, avoidant personality disorder,dependent personality disorder and/or obsessive-compulsive personalitydisorder); Tic disorders (e.g., Tourette's disorder, chronic motor orvocal tic disorder, transient tic disorder and/or tic disorder nototherwise specified); elimination disorders; and any combination of theforegoing as well as any other disorder or group of disorders describedin the Diagnostic and Statistical Manual of Mental Disorders—FourthEdition (DSM-IV; the American Psychiatric Association, Washington D.C.,1994). “Central Nervous System disorders” also include other conditionsthat implicate the CNS including but not limited to neurodegenerativedisorders such as Alzheimer's disease, involuntary movement disorderssuch as Parkinson's disease, Huntington's disease, amyotrophic lateralsclerosis (ALS), and the like. Other CNS disorders include withoutlimitation epilepsy, multiple sclerosis, neurogenic pain, psychogenicpain, and migraines.

In one embodiment, the disorder associated with CNS dysfunction is ademyelinating disease. In one embodiment, the disorder associated withCNS dysfunction is multiple sclerosis, Pelizaeus-Merzbacher disease,Krabbe's disease, metachromatic leukodystrophy, adrenoleukodystrophy,Canavan disease, Alexander disease, orthochromatic leukodystrophy,Zellweger disease, 18q-syndrome, cerebral palsy, spinal cord injury,traumatic brain injury, stroke, phenylketonuria, or viral infection, orany other disorder known or later found to be associated with CNSdysfunction. In another embodiment, the methods of the invention areused to treat a disorder that is not directly associated with CNSdysfunction but would benefit by expression of a heterologouspolypeptide or nucleic acid in CNS cells. Examples include, withoutlimitation, neurodegenerative disorders such as Alzheimer's disease,Parkinson's disease, and Huntington's disease, CNS tumors, and other CNSdisorders.

In other embodiments, the CNS disorder encompasses any subset of theforegoing diseases or excludes any one or more of the foregoingconditions. In particular embodiments, the term “central nervous systemdisorder” does not encompass benign and/or malignant tumors of the CNS.

In certain embodiments, the CNS disorder is Rett Syndrome. In furtherembodiments, the invention relates to a method of treating Rett Syndromein a mammalian subject in need thereof. In certain embodiments, themethod comprises administering a therapeutically effective amount of theAAV particle of the invention, e.g., an AAV particle comprising anucleic acid encoding methyl cytosine binding protein 2.

In another aspect of the invention, the chimeric AAV capsid and vectorsof the invention are fully- or nearly fully-detargeted vectors that canbe further modified to a desirable tropic profile for targeting of oneor more peripheral organs or tissues as discussed below. In this aspect,the present invention also relates to methods for deliveringheterologous nucleotide sequences into a broad range of cells, includingdividing and non-dividing cells. The virus vectors of the invention maybe employed to deliver a nucleotide sequence of interest to a cell invitro, e.g., to produce a polypeptide in vitro or for ex vivo genetherapy. The vectors are additionally useful in a method of delivering anucleotide sequence to a subject in need thereof, e.g., to express atherapeutic or immunogenic polypeptide or nucleic acid. In this manner,the polypeptide or nucleic acid may thus be produced in vivo in thesubject. The subject may be in need of the polypeptide or nucleic acidbecause the subject has a deficiency of the polypeptide, or because theproduction of the polypeptide or nucleic acid in the subject may impartsome therapeutic effect, as a method of treatment or otherwise, and asexplained further below.

In general, the virus vectors of the invention may be employed todeliver any foreign nucleic acid with a biological effect to treat orameliorate the symptoms associated with any disorder related to geneexpression. Further, the invention can be used to treat any diseasestate for which it is beneficial to deliver a therapeutic polypeptide.Illustrative disease states include, but are not limited to: cysticfibrosis (cystic fibrosis transmembrane regulator protein) and otherdiseases of the lung, hemophilia A (Factor VIII), hemophilia B (FactorIX), thalassemia (β-globin), anemia (erythropoietin) and other blooddisorders, Alzheimer's disease (GDF; neprilysin), multiple sclerosis(β-interferon), Parkinson's disease (glial-cell line derivedneurotrophic factor [GDNF]), Huntington's disease (inhibitory RNAincluding without limitation RNAi such as siRNA or shRNA, antisense RNAor microRNA to remove repeats), amyotrophic lateral sclerosis, epilepsy(galanin, neurotrophic factors), and other neurological disorders,cancer (endostatin, angiostatin, TRAIL, FAS-ligand, cytokines includinginterferons; inhibitory RNA including without limitation RNAi (such assiRNA or shRNA), antisense RNA and microRNA including inhibitory RNAagainst VEGF, the multiple drug resistance gene product or a cancerimmunogen), diabetes mellitus (insulin, PGC-α1, GLP-1, myostatinpro-peptide, glucose transporter 4), muscular dystrophies includingDuchenne and Becker (e.g., dystrophin, mini-dystrophin,micro-dystrophin, insulin-like growth factor I, a sarcoglycan [e.g., α,β, γ], Inhibitory RNA [e.g., RNAi, antisense RNA or microRNA] againstmyostatin or myostatin propeptide, laminin-alpha2, Fukutin-relatedprotein, dominant negative myostatin, follistatin, activin type IIsoluble receptor, anti-inflammatory polypeptides such as the Ikappa Bdominant mutant, sarcospan, utrophin, mini-utrophin, inhibitory RNA[e.g., RNAi, antisense RNA or microRNA] against splice junctions in thedystrophin gene to induce exon skipping [see, e.g., WO/2003/095647],inhibitory RNA (e.g., RNAi, antisense RNA or micro RNA] against U7snRNAs to induce exon skipping [see, e.g., WO/2006/021724], andantibodies or antibody fragments against myostatin or myostatinpropeptide), Gaucher disease (glucocerebrosidase), Hurler's disease(α-L-iduronidase), adenosine deaminase deficiency (adenosine deaminase),glycogen storage diseases (e.g., Fabry disease [α-galactosidase] andPompe disease [lysosomal acid α-glucosidase]) and other metabolicdefects including other lysosomal storage disorders and glycogen storagedisorders, congenital emphysema (al-antitrypsin), Lesch-Nyhan Syndrome(hypoxanthine guanine phosphoribosyl transferase), Niemann-Pick disease(sphingomyelinase), Maple Syrup Urine Disease (branched-chain keto aciddehydrogenase), retinal degenerative diseases (and other diseases of theeye and retina; e.g., PDGF, endostatin and/or angiostatin for maculardegeneration), diseases of solid organs such as brain (includingParkinson's Disease [GDNF], astrocytomas [endostatin, angiostatin and/orRNAi against VEGF], glioblastomas [endostatin, angiostatin and/or RNAiagainst VEGF]), liver (RNAi such as siRNA or shRNA, microRNA orantisense RNA for hepatitis B and/or hepatitis C genes), kidney, heartincluding congestive heart failure or peripheral artery disease (PAD)(e.g., by delivering protein phosphatase inhibitor I [I−1],phospholamban, sarcoplasmic endoreticulum Ca²⁺-ATPase [serca2a], zincfinger proteins that regulate the phospholamban gene, Pim-1, PGC-1α,SOD-1, SOD-2, ECF-SOD, kallikrein, thymosin-J34, hypoxia-inducibletranscription factor [HIF], βarkct, β2-adrenergic receptor,β2-adrenergic receptor kinase [βARK], phosphoinositide-3 kinase [PI3kinase], calsarcin, an angiogenic factor, S100A1, parvalbumin, adenylylcyclase type 6, a molecule that effects G-protein coupled receptorkinase type 2 knockdown such as a truncated constitutively activebARKct, an inhibitory RNA [e.g., RNAi, antisense RNA or microRNA]against phospholamban; phospholamban inhibitory or dominant-negativemolecules such as phospholamban S16E, etc.), arthritis (insulin-likegrowth factors), joint disorders (insulin-like growth factors), intimalhyperplasia (e.g., by delivering enos, inos), improve survival of hearttransplants (superoxide dismutase), AIDS (soluble CD4), muscle wasting(insulin-like growth factor I, myostatin pro-peptide, an anti-apoptoticfactor, follistatin), limb ischemia (VEGF, FGF, PGC-1α, EC-SOD, HIF),kidney deficiency (erythropoietin), anemia (erythropoietin), arthritis(anti-inflammatory factors such as TRAP and TNFα soluble receptor),hepatitis (α-interferon), LDL receptor deficiency (LDL receptor),hyperammonemia (ornithine transcarbamylase), spinal cerebral ataxiasincluding SCA1, SCA2 and SCA3, phenylketonuria (phenylalaninehydroxylase), autoimmune diseases, and the like. The invention canfurther be used following organ transplantation to increase the successof the transplant and/or to reduce the negative side effects of organtransplantation or adjunct therapies (e.g., by administeringimmunosuppressant agents or inhibitory nucleic acids to block cytokineproduction). As another example, bone morphogenic proteins (includingRANKL and/or VEGF) can be administered with a bone allograft, forexample, following a break or surgical removal in a cancer patient.

Exemplary lysosomal storage diseases that can be treated according tothe present invention include without limitation: Hurler's Syndrome (MPSScheie's Syndrome (MPS IS), and Hurler-Scheie Syndrome (MPS IH/S)(α-L-iduronidase); Hunter's Syndrome (MPS II) (iduronate sulfatesulfatase); Sanfilippo A Syndrome (MPS IIIA) (Heparan-S-sulfatesulfaminidase), Sanfilippo B Syndrome (MPS IIIB)(N-acetyl-D-glucosaminidase), Sanfilippo C Syndrome (MPS IIIC)(Acetyl-CoA-glucosaminide N-acetyltransferase), Sanfilippo D Syndrome(MPS IIID) (N-acetyl-glucosaminine-6-sulfate sulfatase); Morquio Adisease (MPS IVA) (Galactosamine-6-sulfate sulfatase), Morquio B disease(MPS IV B) (β-Galactosidase); Maroteaux-lmay disease (MPS VI)(arylsulfatase B); Sly Syndrome (MPS VII) (β-glucuronidase);hyaluronidase deficiency (MPS IX) (hyaluronidase); sialidosis(mucolipidosis I), mucolipidosis II (I-Cell disease)(N-actylglucos-aminyl-1-phosphotransferase catalytic subunit),mucolipidosis III (pseudo-Hurler polydystrophy)(N-acetylglucos-aminyl-1-phosphotransferase; type IIIA [catalyticsubunit] and type IIIC [substrate recognition subunit]); GM1gangliosidosis (ganglioside β-galactosidase), GM2 gangliosidosis Type I(Tay-Sachs disease) (β-hexaminidase A), GM2 gangliosidosis type II(Sandhoff's disease) (β-hexosaminidase B); Niemann-Pick disease (Types Aand B) (sphingomyelinase); Gaucher's disease (glucocerebrosidase);Farber's disease (ceraminidase); Fabry's disease (α-galactosidase A);Krabbe's disease (galactosylceramide β-galactosidase); metachromaticleukodystrophy (arylsulfatase A); lysosomal acid lipase deficiencyincluding Wolman's disease (lysosomal acid lipase); Batten disease(juvenile neuronal ceroid lipofuscinosis) (lysosomal trans-membrane CLN3protein) sialidosis (neuraminidase 1); galactosialidosis (Goldberg'ssyndrome) (protective protein/cathepsin A); α-mannosidosis(α-D-mannosidase); β-mannosidosis (β-D-mannosidosis); fucosidosis(α-D-fucosidase); aspartylglucosaminuria (N-Aspartylglucosaminidase);and sialuria (Na phosphate cotransporter).

Exemplary glycogen storage diseases that can be treated according to thepresent invention include, but are not limited to, Type Ia GSD (vonGierke disease) (glucose-6-phosphatase), Type Ib GSD(glucose-6-phosphate translocase), Type Ic GSD (microsomal phosphate orpyrophosphate transporter), Type Id GSD (microsomal glucosetransporter), Type II GSD including Pompe disease or infantile Type IIaGSD (lysosomal acid α-glucosidase) and Type IIb (Danon) (lysosomalmembrane protein-2), Type Ma and IIIb GSD (Debrancher enzyme;amyloglucosidase and oligoglucanotransferase), Type IV GSD (Andersen'sdisease) (branching enzyme), Type V GSD (McArdle disease) (musclephosphorylase), Type VI GSD (Hers' disease) (liver phosphorylase), TypeVII GSD (Tarui's disease) (phosphofructokinase), GSD Type VIII/IXa(X-linked phosphorylase kinase), GSD Type IXb (Liver and musclephosphorylase kinase), GSD Type IXc (liver phosphorylase kinase), GSDType IXd (muscle phosphorylase kinase), GSD 0 (glycogen synthase),Fanconi-Bickel syndrome (glucose transporter-2), phosphoglucoisomerasedeficiency, muscle phosphoglycerate kinase deficiency, phosphoglyceratemutase deficiency, fructose 1,6-diphosphatase deficiency,phosphoenolpyruvate carboxykinase deficiency, and lactate dehydrogenasedeficiency.

Nucleic acids and polypeptides that can be delivered to cardiac muscleinclude those that are beneficial in the treatment of damaged,degenerated or atrophied cardiac muscle and/or congenital cardiacdefects. For example, angiogenic factors useful for facilitatingvascularization in the treatment of heart disease include but are notlimited to vascular endothelial growth factor (VEGF), VEGF II, VEGF-B,VEGF-C, VEGF-D, VEGF-E, VEGF₁₂₁, VEGF₁₃₈, VEGF₁₄₅, VEGF₁₆₅, VEGF₁₈₉,VEGF₂₀₆, hypoxia inducible factor 1α (HIF 1α), endothelial NO synthase(eNOS), iNOS, VEFGR-1 (Flt1), VEGFR-2 (KDR/Flk1), VEGFR-3 (Flt4),angiogenin, epidermal growth factor (EGF), angiopoietin,platelet-derived growth factor, angiogenic factor, transforming growthfactor-α (TGF-α), transforming growth factor-β (TGF-β), vascularpermeability factor (VPF), tumor necrosis factor alpha (TNF-α),interleukin-3 (IL-3), interleukin-8 (IL-8), platelet-derived endothelialgrowth factor (PD-EGF), granulocyte colony stimulating factor (G-CSF),hepatocyte growth factor (HGF), scatter factor (SF), pleitrophin,proliferin, follistatin, placental growth factor (PIGF), midkine,platelet-derived growth factor-BB (PDGF), fractalkine, ICAM-1,angiopoietin-1 and -2 (Ang1 and Ang2), Tie-2, neuropilin-1, ICAM-1,chemokines and cytokines that stimulate smooth muscle cell, monocyte, orleukocyte migration, anti-apoptotic peptides and proteins, fibroblastgrowth factors (FGF), FGF-1, FGF-1b, FGF-1c, FGF-2, FGF-2b, FGF-2c,FGF-3, FGF-3b, FGF-3c, FGF-4, FGF-5, FGF-7, FGF-9, acidic FGF, basicFGF, monocyte chemotactic protein-1, granulocyte macrophage-colonystimulating factor, insulin-like growth factor-1 (IGF-1), IGF-2, earlygrowth response factor-1 (EGR-1), ETS-1, human tissue kallikrein (HK),matrix metalloproteinase, chymase, urokinase-type plasminogen activatorand heparinase. (see, e.g., U.S. Patent Application No. 20060287259 andU.S. Patent Application No. 20070059288).

The most common congenital heart disease found in adults is bicuspidaortic valve, whereas atrial septal defect is responsible for 30-40% ofcongenital heart disease seen in adults. The most common congenitalcardiac defect observed in the pediatric population is ventricularseptal defect. Other congenital heart diseases include Eisenmenger'ssyndrome, patent ductus arteriosus, pulmonary stenosis, coarctation ofthe aorta, transposition of the great arteries, tricuspid atresia,univentricular heart, Ebstein's anomaly, and double-outlet rightventricle. A number of studies have identified putative genetic lociassociated with one or more of these congenital heart diseases. Forexample, the putative gene(s) for congenital heart disease associatedwith Down syndrome is 21q22.2-q22.3, between ETS2 and MX1. Similarly,most cases of DiGeorge syndrome result from a deletion of chromosome22q11.2 (the DiGeorge syndrome chromosome region, or DGCR). Severalgenes are lost in this deletion including the putative transcriptionfactor TUPLE1. This deletion is associated with a variety of phenotypes,e.g., Shprintzen syndrome; conotruncal anomaly face (or Takao syndrome);and isolated outflow tract defects of the heart including Tetralogy ofFallot, truncus arteriosus, and interrupted aortic arch. All of theforegoing disorders can be treated according to the present invention.

Other significant diseases of the heart and vascular system are alsobelieved to have a genetic, typically polygenic, etiological component.These diseases include, for example, hypoplastic left heart syndrome,cardiac valvular dysplasia, Pfeiffer cardiocranial syndrome,oculofaciocardiodental syndrome, Kapur-Toriello syndrome, Sonodasyndrome, Ohdo Blepharophimosis syndrome, heart-hand syndrome,Pierre-Robin syndrome, Hirschsprung disease, Kousseff syndrome, Grangeocclusive arterial syndrome, Kearns-Sayre syndrome, Kartagener syndrome,Alagille syndrome, Ritscher-Schinzel syndrome, Ivemark syndrome,Young-Simpson syndrome, hemochromatosis, Holzgreve syndrome, Barthsyndrome, Smith-Lemli-Opitz syndrome, glycogen storage disease,Gaucher-like disease, Fabry disease, Lowry-Maclean syndrome, Rettsyndrome, Opitz syndrome, Marfan syndrome, Miller-Dieker lissencephalysyndrome, mucopolysaccharidosis, Bruada syndrome, humerospinaldysostosis, Phaver syndrome, McDonough syndrome, Marfanoid hypermobilitysyndrome, atransferrinemia, Cornelia de Lange syndrome, Leopardsyndrome, Diamond-Blackfan anemia, Steinfeld syndrome, progeria, andWilliams-Beuren syndrome. All of these disorders can be treatedaccording to the present invention.

Anti-apoptotic factors can be delivered to skeletal muscle, diaphragmmuscle and/or cardiac muscle to treat muscle wasting diseases, limbischemia, cardiac infarction, heart failure, coronary artery diseaseand/or type I or type II diabetes.

Nucleic acids that can be delivered to skeletal muscle include thosethat are beneficial in the treatment of damaged, degenerated and/oratrophied skeletal muscle. The genetic defects that cause musculardystrophy are known for many forms of the disease. These defective geneseither fail to produce a protein product, produce a protein product thatfails to function properly, or produce a dysfunctional protein productthat interferes with the proper function of the cell. The heterologousnucleic acid may encode a therapeutically functional protein or apolynucleotide that inhibits production or activity of a dysfunctionalprotein. Polypeptides that may be expressed from delivered nucleicacids, or inhibited by delivered nucleic acids (e.g., by deliveringRNAi, microRNA or antisense RNA), include without limitation dystrophin,a mini-dystrophin or a micro-dystrophin (Duchene's and Becker MD);dystrophin-associated glycoproteins (3-sarcoglycan (limb-girdle MD 2E),δ-sarcoglycan (limb-girdle MD 2 2F), α-sarcoglycan (limb girdle MD 2D)and γ-sarcoglycan (limb-girdle MD 2C), utrophin, calpain (autosomalrecessive limb-girdle MD type 2A), caveolin-3 (autosomal-dominantlimb-girdle MD), laminin-alpha2 (merosin-deficient congenital MD),miniagrin (laminin-alpha2 deficient congenital MD), fukutin (Fukuyamatype congenital MD), emerin (Emery-Dreifuss MD), myotilin, lamin A/C,calpain-3, dysferlin, and/or telethonin. Further, the heterologousnucleic acid can encode mir-1, mir-133, mir-206, mir-208 or an antisenseRNA, RNAi (e.g., siRNA or shRNA) or microRNA to induce exon skipping ina defective dystrophin gene.

In particular embodiments, the nucleic acid is delivered to tonguemuscle (e.g., to treat dystrophic tongue). Methods of delivering to thetongue can be by any method known in the art including direct injection,oral administration, topical administration to the tongue, intravenousadministration, intra-articular administration and the like.

The foregoing proteins can also be administered to diaphragm muscle totreat muscular dystrophy.

Alternatively, a gene transfer vector may be administered that encodesany other therapeutic polypeptide.

In particular embodiments, a virus vector according to the presentinvention is used to deliver a nucleic acid of interest as describedherein to skeletal muscle, diaphragm muscle and/or cardiac muscle, forexample, to treat a disorder associated with one or more of thesetissues such as muscular dystrophy, heart disease (including PAD andcongestive heart failure), and the like.

Gene transfer has substantial potential use in understanding andproviding therapy for disease states. There are a number of inheriteddiseases in which defective genes are known and have been cloned. Ingeneral, the above disease states fall into two classes: deficiencystates, usually of enzymes, which are generally inherited in a recessivemanner, and unbalanced states, which may involve regulatory orstructural proteins, and which are typically inherited in a dominantmanner. For deficiency state diseases, gene transfer can be used tobring a normal gene into affected tissues for replacement therapy, aswell as to create animal models for the disease using inhibitory RNAsuch as RNAi (e.g., siRNA or shRNA), microRNA or antisense RNA. Forunbalanced disease states, gene transfer can be used to create a diseasestate in a model system, which can then be used in efforts to counteractthe disease state. Thus, the virus vectors according to the presentinvention permit the treatment of genetic diseases. As used herein, adisease state is treated by partially or wholly remedying the deficiencyor imbalance that causes the disease or makes it more severe. The use ofsite-specific recombination of nucleic sequences to cause mutations orto correct defects is also possible.

The virus vectors according to the present invention may also beemployed to provide an antisense nucleic acid or inhibitory RNA (e.g.,microRNA or RNAi such as a siRNA or shRNA) to a cell in vitro or invivo. Expression of the inhibitory RNA in the target cell diminishesexpression of a particular protein(s) by the cell. Accordingly,inhibitory RNA may be administered to decrease expression of aparticular protein in a subject in need thereof. Inhibitory RNA may alsobe administered to cells in vitro to regulate cell physiology, e.g., tooptimize cell or tissue culture systems.

As a further aspect, the virus vectors of the present invention may beused to produce an immune response in a subject. According to thisembodiment, a virus vector comprising a nucleic acid encoding animmunogen may be administered to a subject, and an active immuneresponse (optionally, a protective immune response) is mounted by thesubject against the immunogen. Immunogens are as described hereinabove.

Alternatively, the virus vector may be administered to a cell ex vivoand the altered cell is administered to the subject. The heterologousnucleic acid is introduced into the cell, and the cell is administeredto the subject, where the heterologous nucleic acid encoding theimmunogen is optionally expressed and induces an immune response in thesubject against the immunogen. In particular embodiments, the cell is anantigen-presenting cell (e.g., a dendritic cell).

An “active immune response” or “active immunity” is characterized by“participation of host tissues and cells after an encounter with theimmunogen. It involves differentiation and proliferation ofimmunocompetent cells in lymphoreticular tissues, which lead tosynthesis of antibody or the development of cell-mediated reactivity, orboth.” Herbert B. Herscowitz, Immunophysiology: Cell Function andCellular Interactions in Antibody Formation, in IMMUNOLOGY: BASICPROCESSES 117 (Joseph A. Bellanti ed., 1985). Alternatively stated, anactive immune response is mounted by the host after exposure toimmunogens by infection or by vaccination. Active immunity can becontrasted with passive immunity, which is acquired through the“transfer of preformed substances (antibody, transfer factor, thymicgraft, interleukin-2) from an actively immunized host to a non-immunehost.” Id.

A “protective” immune response or “protective” immunity as used hereinindicates that the immune response confers some benefit to the subjectin that it prevents or reduces the incidence of disease. Alternatively,a protective immune response or protective immunity may be useful in thetreatment of disease, in particular cancer or tumors (e.g., by causingregression of a cancer or tumor and/or by preventing metastasis and/orby preventing growth of metastatic nodules). The protective effects maybe complete or partial, as long as the benefits of the treatmentoutweigh any disadvantages thereof.

The virus vectors of the present invention may also be administered forcancer immunotherapy by administration of a viral vector expressing acancer cell antigen (or an immunologically similar molecule) or anyother immunogen that produces an immune response against a cancer cell.To illustrate, an immune response may be produced against a cancer cellantigen in a subject by administering a viral vector comprising aheterologous nucleotide sequence encoding the cancer cell antigen, forexample to treat a patient with cancer. The virus vector may beadministered to a subject in vivo or by using ex vivo methods, asdescribed herein.

As used herein, the term “cancer” encompasses tumor-forming cancers.Likewise, the term “cancerous tissue” encompasses tumors. A “cancer cellantigen” encompasses tumor antigens.

The term “cancer” has its understood meaning in the art, for example, anuncontrolled growth of tissue that has the potential to spread todistant sites of the body (i.e., metastasize). Exemplary cancersinclude, but are not limited to, leukemia, lymphoma (e.g., Hodgkin andnon-Hodgkin lymphomas), colorectal cancer, renal cancer, liver cancer,breast cancer, lung cancer, prostate cancer, testicular cancer, ovariancancer, uterine cancer, cervical cancer, brain cancer (e.g., gliomas andglioblastoma), bone cancer, sarcoma, melanoma, head and neck cancer,esophageal cancer, thyroid cancer, and the like. In embodiments of theinvention, the invention is practiced to treat and/or preventtumor-forming cancers.

The term “tumor” is also understood in the art, for example, as anabnormal mass of undifferentiated cells within a multicellular organism.Tumors can be malignant or benign. In representative embodiments, themethods disclosed herein are used to prevent and treat malignant tumors.

Cancer cell antigens have been described hereinabove. By the terms“treating cancer” or “treatment of cancer,” it is intended that theseverity of the cancer is reduced or the cancer is prevented or at leastpartially eliminated. For example, in particular contexts, these termsindicate that metastasis of the cancer is prevented or reduced or atleast partially eliminated. In further representative embodiments theseterms indicate that growth of metastatic nodules (e.g., after surgicalremoval of a primary tumor) is prevented or reduced or at leastpartially eliminated. By the terms “prevention of cancer” or “preventingcancer” it is intended that the methods at least partially eliminate orreduce the incidence or onset of cancer. Alternatively stated, the onsetor progression of cancer in the subject may be slowed, controlled,decreased in likelihood or probability, or delayed.

In particular embodiments, cells may be removed from a subject withcancer and contacted with a virus vector according to the presentinvention. The modified cell is then administered to the subject,whereby an immune response against the cancer cell antigen is elicited.This method is particularly advantageously employed withimmunocompromised subjects that cannot mount a sufficient immuneresponse in vivo (i.e., cannot produce enhancing antibodies insufficient quantities).

It is known in the art that immune responses may be enhanced byimmunomodulatory cytokines (e.g., α-interferon, β-interferon,γ-interferon, ω-interferon, τ-interferon, interleukin-1α,interleukin-1β, interleukin-2, interleukin-3, interleukin-4, interleukin5, interleukin-6, interleukin-7, interleukin-8, interleukin-9,interleukin-10, interleukin-11, interleukin 12, interleukin-13,interleukin-14, interleukin-18, B cell Growth factor, CD40 Ligand, tumornecrosis factor-α, tumor necrosis factor-β, monocyte chemoattractantprotein-1, granulocyte-macrophage colony stimulating factor, andlymphotoxin). Accordingly, immunomodulatory cytokines (e.g., CTLinductive cytokines) may be administered to a subject in conjunctionwith the virus vectors.

Cytokines may be administered by any method known in the art. Exogenouscytokines may be administered to the subject, or alternatively, anucleotide sequence encoding a cytokine may be delivered to the subjectusing a suitable vector, and the cytokine produced in vivo.

The viral vectors are further useful for targeting CNS cells forresearch purposes, e.g., for study of CNS function in vitro or inanimals or for use in creating and/or studying animal models of disease.For example, the vectors can be used to deliver heterologous nucleicacids to neurons in animal models of neural injury, e.g., traumaticbrain injury or spinal cord injury or animal models of neurodegenerativediseases. For example, the vectors can be used to deliver heterologousnucleic acids to oligodendrocytes in animal models of demyelinatingdiseases. Demyelination can be induced in animals by a variety of means,including without limitation administration of viruses (e.g., Semlikivirus, murine hepatitis virus, or Theiler's murine encephalomyelitisvirus) and administration of chemicals (e.g., cuprizone, ethidiumbromide, or lysolecithin). In some embodiments, the vector can also beused in animal models of experimental autoimmune encephalomyelitis. Thiscondition can be induced by, for example, administration of kainite,SIN-1, anti-galactocerebroside, or irradiation. In other embodiments,the viral vector can be used to specifically deliver to oligodendrocytesa toxic agent or an enzyme that produces a toxic agent (e.g., thymidinekinase) in order to kill some or all of the cells.

Further, the virus vectors according to the present invention findfurther use in diagnostic and screening methods, whereby a gene ofinterest is transiently or stably expressed in a cell culture system, oralternatively, a transgenic animal model. The invention can also bepracticed to deliver a nucleic acid for the purposes of proteinproduction, e.g., for laboratory, industrial or commercial purposes.

Recombinant virus vectors according to the present invention find use inboth veterinary and medical applications. Suitable subjects include bothavians and mammals. The term “avian” as used herein includes, but is notlimited to, chickens, ducks, geese, quail, turkeys, pheasant, parrots,parakeets. The term “mammal” as used herein includes, but is not limitedto, humans, primates non-human primates (e.g., monkeys and baboons),cattle, sheep, goats, pigs, horses, cats, dogs, rabbits, rodents (e.g.,rats, mice, hamsters, and the like), etc. Human subjects includeneonates, infants, juveniles, and adults. Optionally, the subject is “inneed of” the methods of the present invention, e.g., because the subjecthas or is believed at risk for a disorder including those describedherein or that would benefit from the delivery of a nucleic acidincluding those described herein. For example, in particularembodiments, the subject has (or has had) or is at risk for ademyelinating disorder or a spinal cord or brain injury. As a furtheroption, the subject can be a laboratory animal and/or an animal model ofdisease.

In particular embodiments, the present invention provides apharmaceutical composition comprising a virus vector of the invention ina pharmaceutically acceptable carrier and, optionally, other medicinalagents, pharmaceutical agents, stabilizing agents, buffers, carriers,adjuvants, diluents, etc. For injection, the carrier will typically be aliquid. For other methods of administration, the carrier may be eithersolid or liquid. For inhalation administration, the carrier will berespirable, and will preferably be in solid or liquid particulate form.

By “pharmaceutically acceptable” it is meant a material that is nottoxic or otherwise undesirable, i.e., the material may be administeredto a subject without causing any undesirable biological effects.

One aspect of the present invention is a method of transferring anucleotide sequence to a cell in vitro. The virus vector may beintroduced to the cells at the appropriate multiplicity of infectionaccording to standard transduction methods appropriate for theparticular target cells. Titers of the virus vector or capsid toadminister can vary, depending upon the target cell type and number, andthe particular virus vector or capsid, and can be determined by those ofskill in the art without undue experimentation. In particularembodiments, at least about 10³ infectious units, more preferably atleast about 10⁵ infectious units are introduced to the cell.

The cell(s) into which the virus vector can be introduced may be of anytype, including but not limited to neural cells (including cells of theperipheral and central nervous systems, in particular, brain cells suchas neurons, oligodendrocytes, glial cells, astrocytes), lung cells,cells of the eye (including retinal cells, retinal pigment epithelium,and corneal cells), epithelial cells (e.g., gut and respiratoryepithelial cells), skeletal muscle cells (including myoblasts, myotubesand myofibers), diaphragm muscle cells, dendritic cells, pancreaticcells (including islet cells), hepatic cells, a cell of thegastrointestinal tract (including smooth muscle cells, epithelialcells), heart cells (including cardiomyocytes), bone cells (e.g., bonemarrow stem cells), hematopoietic stem cells, spleen cells,keratinocytes, fibroblasts, endothelial cells, prostate cells, jointcells (including, e.g., cartilage, meniscus, synovium and bone marrow),germ cells, and the like. Alternatively, the cell may be any progenitorcell. As a further alternative, the cell can be a stem cell (e.g.,neural stem cell, liver stem cell). As still a further alternative, thecell may be a cancer or tumor cell (cancers and tumors are describedabove). Moreover, the cells can be from any species of origin, asindicated above.

The virus vectors may be introduced to cells in vitro for the purpose ofadministering the modified cell to a subject. In particular embodiments,the cells have been removed from a subject, the virus vector isintroduced therein, and the cells are then replaced back into thesubject. Methods of removing cells from subject for treatment ex vivo,followed by introduction back into the subject are known in the art(see, e.g., U.S. Pat. No. 5,399,346). Alternatively, the recombinantvirus vector is introduced into cells from another subject, intocultured cells, or into cells from any other suitable source, and thecells are administered to a subject in need thereof.

Suitable cells for ex vivo gene therapy are as described above. Dosagesof the cells to administer to a subject will vary upon the age,condition and species of the subject, the type of cell, the nucleic acidbeing expressed by the cell, the mode of administration, and the like.Typically, at least about 10² to about 10⁸ or about 10³ to about 10⁶cells will be administered per dose in a pharmaceutically acceptablecarrier. In particular embodiments, the cells transduced with the virusvector are administered to the subject in an effective amount incombination with a pharmaceutical carrier.

In some embodiments, cells that have been transduced with the virusvector may be administered to elicit an immunogenic response against thedelivered polypeptide (e.g., expressed as a transgene or in the capsid).Typically, a quantity of cells expressing an effective amount of thepolypeptide in combination with a pharmaceutically acceptable carrier isadministered. Optionally, the dosage is sufficient to produce aprotective immune response (as defined above). The degree of protectionconferred need not be complete or permanent, as long as the benefits ofadministering the immunogenic polypeptide outweigh any disadvantagesthereof.

A further aspect of the invention is a method of administering the virusvectors or capsids of the invention to subjects. In particularembodiments, the method comprises a method of delivering a nucleic acidof interest to an animal subject, the method comprising: administeringan effective amount of a virus vector according to the invention to ananimal subject. Administration of the virus vectors of the presentinvention to a human subject or an animal in need thereof can be by anymeans known in the art. Optionally, the virus vector is delivered in aneffective dose in a pharmaceutically acceptable carrier.

The virus vectors of the invention can further be administered to asubject to elicit an immunogenic response (e.g., as a vaccine).Typically, vaccines of the present invention comprise an effectiveamount of virus in combination with a pharmaceutically acceptablecarrier. Optionally, the dosage is sufficient to produce a protectiveimmune response (as defined above). The degree of protection conferredneed not be complete or permanent, as long as the benefits ofadministering the immunogenic polypeptide outweigh any disadvantagesthereof. Subjects and immunogens are as described above.

Dosages of the virus vectors to be administered to a subject will dependupon the mode of administration, the disease or condition to be treated,the individual subject's condition, the particular virus vector, and thenucleic acid to be delivered, and can be determined in a routine manner.Exemplary doses for achieving therapeutic effects are virus titers of atleast about 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³, 10¹⁴, 10¹⁵transducing units or more, preferably about 10⁷ or 10⁸, 10⁹, 10¹⁰, 10¹¹,10¹², 10¹³ or 10¹⁴ transducing units, yet more preferably about 10¹²transducing units.

In particular embodiments, more than one administration (e.g., two,three, four or more administrations) may be employed to achieve thedesired level of gene expression over a period of various intervals,e.g., daily, weekly, monthly, yearly, etc.

Exemplary modes of administration include oral, rectal, transmucosal,topical, intranasal, inhalation (e.g., via an aerosol), buccal (e.g.,sublingual), vaginal, intrathecal, intraocular, transdermal, in utero(or in ovo), parenteral (e.g., intravenous, subcutaneous, intradermal,intramuscular [including administration to skeletal, diaphragm and/orcardiac muscle], intradermal, intrapleural, intracerebral, andintraarticular), topical (e.g., to both skin and mucosal surfaces,including airway surfaces, and transdermal administration),intro-lymphatic, and the like, as well as direct tissue or organinjection (e.g., to liver, skeletal muscle, cardiac muscle, diaphragmmuscle or brain). Administration can also be to a tumor (e.g., in or anear a tumor or a lymph node). The most suitable route in any given casewill depend on the nature and severity of the condition being treatedand on the nature of the particular vector that is being used.

In some embodiments, the viral vector is administered directly to theCNS, e.g., the brain or the spinal cord. Direct administration canresult in high specificity of transduction of CNS cells, e.g., whereinat least 80%, 85%, 90%, 95% or more of the transduced cells are CNScells. Any method known in the art to administer vectors directly to theCNS can be used. The vector may be introduced into the spinal cord,brainstem (medulla oblongata, pons), midbrain (hypothalamus, thalamus,epithalamus, pituitary gland, substantia nigra, pineal gland),cerebellum, telencephalon (corpus striatum, cerebrum including theoccipital, temporal, parietal and frontal lobes, cortex, basal ganglia,hippocampus and amygdala), limbic system, neocortex, corpus striatum,cerebrum, and inferior colliculus. The vector may also be administeredto different regions of the eye such as the retina, cornea or opticnerve. The vector may be delivered into the cerebrospinal fluid (e.g.,by lumbar puncture) for more disperse administration of the vector.

The delivery vector may be administered to the desired region(s) of theCNS by any route known in the art, including but not limited to,intrathecal, intracerebral, intraventricular, intranasal, intra-aural,intra-ocular (e.g., intra-vitreous, sub-retinal, anterior chamber) andperi-ocular (e.g., sub-Tenon's region) delivery or any combinationthereof.

Typically, the viral vector will be administered in a liquid formulationby direct injection (e.g., stereotactic injection) to the desired regionor compartment in the CNS. In some embodiments, the vector can bedelivered via a reservoir and/or pump. In other embodiments, the vectormay be provided by topical application to the desired region or byintra-nasal administration of an aerosol formulation. Administration tothe eye or into the ear, may be by topical application of liquiddroplets. As a further alternative, the vector may be administered as asolid, slow-release formulation. Controlled release of parvovirus andAAV vectors is described by international patent publication WO01/91803.

In some embodiments where the subject has a compromised blood-brainbarrier (BBB), the viral vector can be delivered systemically (e.g.,intravenously) to the subject, wherein the vector transduces CNS cellsin the area of (e.g., bordering) the BBB compromise. In certainembodiments, the vector transduces cells in the compromised area but notcells in uncompromised areas. Thus, one aspect of the invention relatesto a method of delivering a nucleic acid of interest to an area of theCNS bordering a compromised blood brain barrier area in a mammaliansubject, the method comprising intravenously administering an effectiveamount of the AAV particle of the invention.

In some embodiments, the compromise in the BBB is due to a disease ordisorder. Examples include, without limitation, neurodegenerativediseases such as Alzheimer's, Parkinson's disease, disease, amyotrophiclateral sclerosis, and multiple sclerosis, epilepsy, CNS tumors, orcerebral infarcts. In other embodiments, the BBB compromise can be aninduced disruption, e.g., to promote delivery of agents to the CNS.Temporary BBB compromises can be induced by, for example, toxicchemicals (such as metrazol, VP-16, cisplatin, hydroxyurea,fluorouracil, and etoposide), osmotic agents (such as mannitol andarabinose), biological agents (such as retinoic acid, phorbol myristateacetate, leukotriene C4, bradykinin, histamine, RMP-7, andalkylglycerols), or irradiation (such as ultrasound or electromagneticradiation).

Administration to skeletal muscle according to the present inventionincludes but is not limited to administration to skeletal muscles in thelimbs (e.g., upper arm, lower arm, upper leg, and/or lower leg), back,neck, head (e.g., tongue), thorax, abdomen, pelvis/perineum, and/ordigits. Suitable skeletal muscle tissues include but are not limited toabductor digiti minimi (in the hand), abductor digiti minimi (in thefoot), abductor hallucis, abductor ossis metatarsi quinti, abductorpollicis brevis, abductor pollicis longus, adductor brevis, adductorhallucis, adductor longus, adductor magnus, adductor pollicis, anconeus,anterior scalene, articularis genus, biceps brachii, biceps femoris,brachialis, brachioradialis, buccinator, coracobrachialis, corrugatorsupercilii, deltoid, depressor anguli oris, depressor labii inferioris,digastric, dorsal interossei (in the hand), dorsal interossei (in thefoot), extensor carpi radialis brevis, extensor carpi radialis longus,extensor carpi ulnaris, extensor digiti minimi, extensor digitorum,extensor digitorum brevis, extensor digitorum longus, extensor hallucisbrevis, extensor hallucis longus, extensor indicis, extensor pollicisbrevis, extensor pollicis longus, flexor carpi radialis, flexor carpiulnaris, flexor digiti minimi brevis (in the hand), flexor digiti minimibrevis (in the foot), flexor digitorum brevis, flexor digitorum longus,flexor digitorum profundus, flexor digitorum superficialis, flexorhallucis brevis, flexor hallucis longus, flexor pollicis brevis, flexorpollicis longus, frontalis, gastrocnemius, geniohyoid, gluteus maximus,gluteus medius, gluteus minimus, gracilis, iliocostalis cervicis,iliocostalis lumborum, iliocostalis thoracis, illiacus, inferiorgemellus, inferior oblique, inferior rectus, infraspinatus,interspinalis, intertransversi, lateral pterygoid, lateral rectus,latissimus dorsi, levator anguli oris, levator labii superioris, levatorlabii superioris alaeque nasi, levator palpebrae superioris, levatorscapulae, long rotators, longissimus capitis, longissimus cervicis,longissimus thoracis, longus capitis, longus colli, lumbricals (in thehand), lumbricals (in the foot), masseter, medial pterygoid, medialrectus, middle scalene, multifidus, mylohyoid, obliquus capitisinferior, obliquus capitis superior, obturator externus, obturatorinternus, occipitalis, omohyoid, opponens digiti minimi, opponenspollicis, orbicularis oculi, orbicularis oris, palmar interossei,palmaris brevis, palmaris longus, pectineus, pectoralis major,pectoralis minor, peroneus brevis, peroneus longus, peroneus tertius,piriformis, plantar interossei, plantaris, platysma, popliteus,posterior scalene, pronator quadratus, pronator teres, psoas major,quadratus femoris, quadratus plantae, rectus capitis anterior, rectuscapitis lateralis, rectus capitis posterior major, rectus capitisposterior minor, rectus femoris, rhomboid major, rhomboid minor,risorius, sartorius, scalenus minimus, semimembranosus, semispinaliscapitis, semispinalis cervicis, semispinalis thoracis, semitendinosus,serratus anterior, short rotators, soleus, spinalis capitis, spinaliscervicis, spinalis thoracis, splenius capitis, splenius cervicis,sternocleidomastoid, sternohyoid, sternothyroid, stylohyoid, subclavius,subscapularis, superior gemellus, superior oblique, superior rectus,supinator, supraspinatus, temporalis, tensor fascia lata, teres major,teres minor, thoracis, thyrohyoid, tibialis anterior, tibialisposterior, trapezius, triceps brachii, vastus intermedius, vastuslateralis, vastus medialis, zygomaticus major, and zygomaticus minor andany other suitable skeletal muscle as known in the art.

The virus vector can be delivered to skeletal muscle by any suitablemethod including without limitation intravenous administration,intra-arterial administration, intraperitoneal administration, isolatedlimb perfusion (of leg and/or arm; see, e.g. Arruda et al., (2005) Blood105:3458-3464), and/or direct intramuscular injection.

Administration to cardiac muscle includes without limitationadministration to the left atrium, right atrium, left ventricle, rightventricle and/or septum. The virus vector can be delivered to cardiacmuscle by any method known in the art including, e.g., intravenousadministration, intra-arterial administration such as intra-aorticadministration, direct cardiac injection (e.g., into left atrium, rightatrium, left ventricle, right ventricle), and/or coronary arteryperfusion.

Administration to diaphragm muscle can be by any suitable methodincluding intravenous administration, intra-arterial administration,and/or intra-peritoneal administration.

Delivery to any of these tissues can also be achieved by delivering adepot comprising the virus vector, which can be implanted into theskeletal, cardiac and/or diaphragm muscle tissue or the tissue can becontacted with a film or other matrix comprising the virus vector.Examples of such implantable matrices or substrates are described inU.S. Pat. No. 7,201,898).

In particular embodiments, a virus vector according to the presentinvention is administered to skeletal muscle, diaphragm muscle and/orcardiac muscle (e.g., to treat muscular dystrophy or heart disease [forexample, PAD or congestive heart failure]).

The invention can be used to treat disorders of skeletal, cardiac and/ordiaphragm muscle. Alternatively, the invention can be practiced todeliver a nucleic acid to skeletal, cardiac and/or diaphragm muscle,which is used as a platform for production of a protein product (e.g.,an enzyme) or non-translated RNA (e.g., RNAi, microRNA, antisense RNA)that normally circulates in the blood or for systemic delivery to othertissues to treat a disorder (e.g., a metabolic disorder, such asdiabetes (e.g., insulin), hemophilia (e.g., Factor IX or Factor VIII),or a lysosomal storage disorder (such as Gaucher's disease[glucocerebrosidase], Pompe disease [lysosomal acid α-glucosidase] orFabry disease [α-galactosidase A]) or a glycogen storage disorder (suchas Pompe disease [lysosomal acid α glucosidase]). Other suitableproteins for treating metabolic disorders are described above.

In a representative embodiment, the invention provides a method oftreating muscular dystrophy in a subject in need thereof, the methodcomprising: administering an effective amount of a virus vector of theinvention to a mammalian subject, wherein the virus vector comprises aheterologous nucleic acid effective to treat muscular dystrophy. In anexemplary embodiment, the method comprises: administering an effectiveamount of a virus vector of the invention to a mammalian subject,wherein the virus vector comprises a heterologous nucleic acid encodingdystrophin, a mini-dystrophin, a micro-dystrophin, utrophin,mini-utrophin, laminin-α2, mini-agrin, Fukutin-related protein,follistatin, dominant negative myostatin, α-sarcoglycan, β-sarcoglycan,γ-sarcoglycan, δ-sarcoglycan, IGF-1, myostatin pro-peptide, activin typeII soluble receptor, anti-inflammatory polypeptides such as the Ikappa Bdominant mutant, sarcospan, antibodies or antibody fragments againstmyostatin or myostatin propeptide, or an inhibitory RNA (e.g., antisenseRNA, microRNA or RNAi) against myostatin, mir-1, mir-133, mir-206,mir-208 or an inhibitory RNA (e.g., microRNA, RNAi or antisense RNA) toinduce exon skipping in a defective dystrophin gene. In particularembodiments, the virus vector can be administered to skeletal, diaphragmand/or cardiac muscle as described elsewhere herein.

The invention further encompasses a method of treating a metabolicdisorder in a subject in need thereof. In representative embodiments,the method comprises: administering an effective amount of a virusvector of the invention to skeletal muscle of a subject, wherein thevirus vector comprises a heterologous nucleic acid encoding apolypeptide, wherein the metabolic disorder is a result of a deficiencyand/or defect in the polypeptide. Illustrative metabolic disorders andheterologous nucleic acids encoding polypeptides are described herein.As a further option, the heterologous nucleic acid can encode a secretedprotein.

The invention can also be practiced to produce inhibitory RNA (e.g.,antisense RNA, microRNA or RNAi) for systemic delivery.

The invention also provides a method of treating congenital heartfailure in a subject in need thereof, the method comprisingadministering an effective amount of a virus vector of the invention toa mammalian subject, wherein the virus vector comprises a heterologousnucleic acid effective to treat congenital heart failure. Inrepresentative embodiments, the method comprises administering aneffective amount of a virus vector of the invention to a mammaliansubject, wherein the virus vector comprises a heterologous nucleic acidencoding a sarcoplasmic endoreticulum Ca²⁺-ATPase (SERCA2a), anangiogenic factor, phospholamban, PI3 kinase, calsarcan, a β-adrenergicreceptor kinase (βARK), βARKct, inhibitor 1 of protein phosphatase 1,Pim-1, PGC-1α, SOD-1, SOD-2, EC-SOD, Kallikrein, HIF, thymosin-β4,S100A1, parvalbumin, adenylyl cyclase type 6, a molecule that effectsG-protein coupled receptor kinase type 2 knockdown such as a truncatedconstitutively active bARKct; phospholamban inhibitory ordominant-negative molecules such as phospholamban S16E, mir-1, mir-133,mir-206, mir-208.

Injectables can be prepared in conventional forms, either as liquidsolutions or suspensions, solid forms suitable for solution orsuspension in liquid prior to injection, or as emulsions. Alternatively,one may administer the virus vector in a local rather than systemicmanner, for example, in a depot or sustained-release formulation.Further, the virus vector can be delivered dried to a surgicallyimplantable matrix such as a bone graft substitute, a suture, a stent,and the like (e.g., as described in U.S. Pat. No. 7,201,898).

Pharmaceutical compositions suitable for oral administration can bepresented in discrete units, such as capsules, cachets, lozenges, ortablets, each containing a predetermined amount of the composition ofthis invention; as a powder or granules; as a solution or a suspensionin an aqueous or non-aqueous liquid; or as an oil-in-water orwater-in-oil emulsion. Oral delivery can be performed by complexing avirus vector of the present invention to a carrier capable ofwithstanding degradation by digestive enzymes in the gut of an animal.Examples of such carriers include plastic capsules or tablets, as knownin the art. Such formulations are prepared by any suitable method ofpharmacy, which includes the step of bringing into association thecomposition and a suitable carrier (which may contain one or moreaccessory ingredients as noted above). In general, the pharmaceuticalcomposition according to embodiments of the present invention areprepared by uniformly and intimately admixing the composition with aliquid or finely divided solid carrier, or both, and then, if necessary,shaping the resulting mixture. For example, a tablet can be prepared bycompressing or molding a powder or granules containing the composition,optionally with one or more accessory ingredients. Compressed tabletsare prepared by compressing, in a suitable machine, the composition in afree-flowing form, such as a powder or granules optionally mixed with abinder, lubricant, inert diluent, and/or surface active/dispersingagent(s). Molded tablets are made by molding, in a suitable machine, thepowdered compound moistened with an inert liquid binder.

Pharmaceutical compositions suitable for buccal (sub-lingual)administration include lozenges comprising the composition of thisinvention in a flavored base, usually sucrose and acacia or tragacanth;and pastilles comprising the composition in an inert base such asgelatin and glycerin or sucrose and acacia.

Pharmaceutical compositions suitable for parenteral administration cancomprise sterile aqueous and non-aqueous injection solutions of thecomposition of this invention, which preparations are optionallyisotonic with the blood of the intended recipient. These preparationscan contain anti-oxidants, buffers, bacteriostats and solutes, whichrender the composition isotonic with the blood of the intendedrecipient. Aqueous and non-aqueous sterile suspensions, solutions andemulsions can include suspending agents and thickening agents. Examplesof non-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's, or fixedoils. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives may also be present such as,for example, antimicrobials, anti-oxidants, chelating agents, and inertgases and the like.

The compositions can be presented in unit/dose or multi-dose containers,for example, in sealed ampoules and vials, and can be stored in afreeze-dried (lyophilized) condition requiring only the addition of thesterile liquid carrier, for example, saline or water-for-injectionimmediately prior to use.

Extemporaneous injection solutions and suspensions can be prepared fromsterile powders, granules and tablets of the kind previously described.For example, an injectable, stable, sterile composition of thisinvention in a unit dosage form in a sealed container can be provided.The composition can be provided in the form of a lyophilizate, which canbe reconstituted with a suitable pharmaceutically acceptable carrier toform a liquid composition suitable for injection into a subject. Theunit dosage form can be from about 1 μg to about 10 grams of thecomposition of this invention. When the composition is substantiallywater-insoluble, a sufficient amount of emulsifying agent, which isphysiologically acceptable, can be included in sufficient quantity toemulsify the composition in an aqueous carrier. One such usefulemulsifying agent is phosphatidyl choline.

Pharmaceutical compositions suitable for rectal administration can bepresented as unit dose suppositories. These can be prepared by admixingthe composition with one or more conventional solid carriers, such asfor example, cocoa butter and then shaping the resulting mixture.

Pharmaceutical compositions of this invention suitable for topicalapplication to the skin can take the form of an ointment, cream, lotion,paste, gel, spray, aerosol, or oil. Carriers that can be used include,but are not limited to, petroleum jelly, lanoline, polyethylene glycols,alcohols, transdermal enhancers, and combinations of two or morethereof. In some embodiments, for example, topical delivery can beperformed by mixing a pharmaceutical composition of the presentinvention with a lipophilic reagent (e.g., DMSO) that is capable ofpassing into the skin.

Pharmaceutical compositions suitable for transdermal administration canbe in the form of discrete patches adapted to remain in intimate contactwith the epidermis of the subject for a prolonged period of time.Compositions suitable for transdermal administration can also bedelivered by iontophoresis (see, for example, Pharm. Res. 3:318 (1986))and typically take the form of an optionally buffered aqueous solutionof the composition of this invention. Suitable formulations can comprisecitrate or bis\tris buffer (pH 6) or ethanol/water and can contain from0.1 to 0.2M active ingredient.

The virus vectors disclosed herein may be administered to the lungs of asubject by any suitable means, for example, by administering an aerosolsuspension of respirable particles comprised of the virus vectors, whichthe subject inhales. The respirable particles may be liquid or solid.Aerosols of liquid particles comprising the virus vectors may beproduced by any suitable means, such as with a pressure-driven aerosolnebulizer or an ultrasonic nebulizer, as is known to those of skill inthe art. See, e.g., U.S. Pat. No. 4,501,729. Aerosols of solid particlescomprising the virus vectors may likewise be produced with any solidparticulate medicament aerosol generator, by techniques known in thepharmaceutical art.

IV. Use of the AAV Capsid to Target Peripheral Tissues

The AAV capsids and vectors of the present invention have beendemonstrated to be fully or nearly fully detargeted for peripheralorgans and tissues. This detargeting makes the vectors ideal as a“blank” vector that can be altered to produce the desired tropicprofile, e.g., to target specific organs and tissues and/or detargetother organs and tissues. Thus, one aspect of the invention relates to amethod of preparing an AAV capsid having a tropism profile of interest,the method comprising modifying the AAV capsid of the present inventionto insert an amino acid sequence providing the tropism profile ofinterest. In some embodiments, the tropism profile of interest isenhanced selectivity for a tissue selected from skeletal muscle, cardiacmuscle, diaphragm, kidney, liver, pancreas, spleen, gastrointestinaltract, lung, joint tissue, tongue, ovary, testis, a germ cell, a cancercell, or a combination thereof and/or reduced selectivity for a tissueselected from liver, ovary, testis, a germ cell, or a combinationthereof.

Examples of specific targeting and detargeting sequences are known inthe art. One example is the molecular basis for preferential livertropism, which has been mapped, in the case of AAV2 and AAV6, to acontinuous basic footprint that appears to be involved in theinteraction of either serotype with heparin. Specifically, it haspreviously been demonstrated that a single lysine residue on AAV6 (K531)dictates heparin binding ability and consequently, liver tropism. Incorollary, substitutional mutagenesis of the correspondingglutamate/aspartate residue on other serotypes with a lysine residueconfers heparin binding, possibly by forming a minimum continuous basicfootprint on the capsid surface. Another example is the capsid mutantscomprising alterations in the three-fold axis loop 4 as disclosed inInternational Publication No. WO 2012/093784, incorporated herein byreference in its entirety. These mutants exhibit one or more propertiesincluding (i) reduced transduction of liver, (ii) enhanced movementacross endothelial cells, (iii) systemic transduction; (iv) enhancedtransduction of muscle tissue (e.g., skeletal muscle, cardiac muscleand/or diaphragm muscle), and/or (v) reduced transduction of braintissues (e.g., neurons). Other tropic sequences are described in Li etal., (2012) J. Virol. 86:7752-7759; Pulicherla et al., (2011) Mol. Ther.19:1070-1078; Bowles et al., (2012) Mol. Ther. 20:443-455; Asokan etal., (2012) Mol. Ther. 20:699-708; and Asokan et al., (2010) NatureBiotechnol. 28:79-82; each incorporated by reference in its entirety.

In some embodiments, the AAV capsid of the present invention can bemodified through DNA scrambling and/or directed evolution to identifymodified capsids having the desired tropism profile. Techniques for DNAscrambling and directed evolution of AAV capsids are described inInternational Publication No. WO 2009/137006, incorporated herein byreference in its entirety.

V. Chimeric AAV Capsids Targeted to Oligodendrocytes

The inventors have identified chimeric AAV capsid structures capable ofpreferentially transducing oligodendrocytes over neurons and other cellsof the CNS. Thus, one aspect of the invention relates to a nucleic acidencoding an AAV capsid, the nucleic acid comprising, consistingessentially of, or consisting of the VP1, VP2, or VP1/VP2 encodingportion of an AAV capsid coding sequence that is at least 90% identicalto: (a) the nucleotide sequence of SEQ ID NO:129 (BNP61); or (b) anucleotide sequence encoding SEQ ID NO:130 (BNP61); operably linked tothe VP3 portion of a different AAV capsid coding sequence; and virusescomprising the chimeric AAV capsids. In some embodiments, the VP1, VP2,or VP1/VP2 portion of the AAV capsid coding sequence is at least 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the VP1, VP2, orVP1/VP2 encoding portion of the nucleotide sequence of (a) or (b). Inanother embodiment, the AAV capsid coding sequence comprises, consistsessentially of, or consists of the VP1, VP2, or VP1/VP2 encoding portionof the nucleotide sequence of (a) or (b). In some embodiments, the VP3encoding portion of a different AAV capsid coding sequence is awild-type capsid sequence (e.g., AAV8 or AAV9) or a chimeric sequencethat is different from any of the capsid sequences of the presentinvention.

In certain embodiments, the nucleic acid of the invention furtherencodes an E532K substitution in the capsid protein (numbering relativeto the AAV8 capsid sequence).

In some embodiments, the nucleic acid encoding an AAV capsid comprises,consists essentially of, or consists of the VP1, VP2, or VP1/VP2encoding portion of an AAV capsid coding sequence that is at least 90%identical to a nucleotide sequence encoding SEQ ID NOS:131 (BNP62) or132 (BNP63) operably linked to the VP3 encoding portion of a differentAAV capsid sequence; and viruses comprising the chimeric AAV capsids. Insome embodiments, the VP1, VP2, or VP1/VP2 encoding portion of the AAVcapsid coding sequence is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%,98% or 99% identical to the VP1, VP2, or VP1/VP2 encoding portion of thenucleotide sequence encoding SEQ ID NOS:131 or 132. In anotherembodiment, the AAV capsid coding sequence comprises, consistsessentially of, or consists of the VP1, VP2, or VP1/VP2 encoding portionof the nucleotide sequence encoding SEQ ID NOS:131 or 132 operablylinked to the VP3 portion of a different AAV capsid coding sequence. Insome embodiments, the VP3 encoding portion of a different AAV capsidcoding sequence is a wild-type capsid sequence (e.g., AAV8 or AAV9) or achimeric sequence that is different from any of the capsid sequences ofthe present invention.

In certain embodiments, the nucleic acid of the invention furtherencodes an E532K substitution in the capsid protein (numbering relativeto the AAV8 capsid sequence).

SEQ ID NOS:130-132 show examples of the VP1 capsid protein sequences ofthe invention. The designation of all amino acid positions in thedescription of the invention and the appended claims is with respect toVP1 numbering. Those skilled in the art will understand that the AAVcapsid generally contains the smaller VP2 and VP3 capsid proteins aswell. Due to the overlap of the coding sequences for the AAV capsidproteins, the nucleic acid coding sequences and amino acid sequences ofthe VP2 and VP3 capsid proteins will be apparent from the VP1 sequencesshown in SEQ ID NOS:129-132. In particular, VP2 starts at nucleotide 412(acg) of SEQ ID NO:129 and threonine 148 of SEQ ID NO:130. VP3 starts atnucleotide 607 (atg) of SEQ ID NO:129 and methionine 203 of SEQ IDNO:130. In certain embodiments, isolated VP2 and VP3 capsid proteinscomprising the sequence from SEQ ID NOS:130-132 and isolated nucleicacids encoding the VP2 or VP3 proteins, or both, are contemplated.

In certain embodiments, the capsid of the invention further comprises anE532K substitution (numbering relative to the AAV8 capsid sequence).

The invention also provides chimeric AAV capsid proteins and chimericcapsids, wherein the capsid protein comprises, consists essentially of,or consists of an amino acid sequence as shown in one of SEQ IDNOS:130-132, wherein 1, 2 or fewer, 3 or fewer, 4 or fewer, 5 or fewer,6 or fewer, 7 or fewer, 8 or fewer, 9 or fewer, 10 or fewer, 12 orfewer, 15 or fewer, 20 or fewer, 25 or fewer, 30 or fewer, 40 or fewer,or 50 or fewer of the amino acids within the capsid protein codingsequence of one of SEQ ID NOS:130-132 is substituted by another aminoacid (naturally occurring, modified and/or synthetic), optionally aconservative amino acid substitution, and/or are deleted and/or thereare insertions (including N-terminal and C-terminal extensions) of 1, 2or fewer, 3 or fewer, 4 or fewer, 5 or fewer, 6 or fewer, 7 or fewer, 8or fewer, 9 or fewer, 10 or fewer, 12 or fewer, 15 or fewer, 20 orfewer, 25 or fewer, 30 or fewer, 40 or fewer, or 50 or fewer amino acidsor any combination of substitutions, deletions and/or insertions,wherein the substitutions, deletions and/or insertions do not undulyimpair the structure and/or function of a virion (e.g., an AAV virion)comprising the variant capsid protein or capsid. For example, inrepresentative embodiments of the invention, an AAV virion comprisingthe chimeric capsid protein substantially retains at least one propertyof a chimeric virion comprising a chimeric capsid protein as shown inone of SEQ ID NOS:130-132. For example, the virion comprising thechimeric capsid protein can substantially retain the oligodendrocytetropism profile of a virion comprising the chimeric AAV capsid proteinas shown in one of SEQ ID NOS:130-132. Methods of evaluating biologicalproperties such as virus transduction are well-known in the art (see,e.g., the Examples).

A further embodiment of the invention relates to a nucleic acid encodingan AAV8 capsid, the capsid comprising an E532K substitution. In someembodiments, the nucleic acid comprises, consists essentially of, orconsists of an AAV capsid coding sequence that is at least 90% identicalto: (a) the nucleotide sequence of SEQ ID NO:133 (AAV8 E532K capsidnucleotide sequence); or (b) a nucleotide sequence encoding SEQ IDNO:134 (AAV8 E532K capsid amino acid sequence); and viruses comprisingthe chimeric AAV capsids. In some embodiments, the AAV capsid codingsequence is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%identical to the nucleotide sequence of (a) or (b). In anotherembodiment, the AAV capsid coding sequence comprises, consistsessentially of, or consists of the nucleotide sequence of (a) or (b). Insome embodiments, the AAV8 E532K capsid coding sequence furthercomprises the VP1, VP2, or VP1/VP2 encoding portions of the capsidssequences of the invention.

Conservative amino acid substitutions are known in the art. Inparticular embodiments, a conservative amino acid substitution includessubstitutions within one or more of the following groups: glycine,alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid;asparagine, glutamine; serine, threonine; lysine, arginine; and/orphenylalanine, tyrosine.

It will be apparent to those skilled in the art that the amino acidsequences of the chimeric AAV capsid protein of SEQ ID NOS:130-132and/or the AAV8 E532K substitution can further be modified toincorporate other modifications as known in the art to impart desiredproperties. As nonlimiting possibilities, the capsid protein can bemodified to incorporate targeting sequences (e.g., RGD) or sequencesthat facilitate purification and/or detection. For example, the capsidprotein can be fused to all or a portion of glutathione-S-transferase,maltose-binding protein, a heparin/heparan sulfate binding domain,poly-His, a ligand, and/or a reporter protein (e.g., Green FluorescentProtein, β-glucuronidase, β-galactosidase, luciferase, etc.), animmunoglobulin Fc fragment, a single-chain antibody, hemagglutinin,c-myc, FLAG epitope, and the like to form a fusion protein. Methods ofinserting targeting peptides into the AAV capsid are known in the art(see, e.g., international patent publication WO 00/28004; Nicklin etal., (2001) Mol. Ther. 474-181; White et al., (2004) Circulation109:513-319; Muller et al., (2003) Nature Biotech. 21:1040-1046.

The viruses of the invention can further comprise a duplexed viralgenome as described in international patent publication WO 01/92551 andU.S. Pat. No. 7,465,583.

The invention also provides AAV capsids comprising the chimeric AAVcapsid proteins of the invention and virus particles (i.e., virions)comprising the same, wherein the virus particle packages (i.e.,encapsidates) a vector genome, optionally an AAV vector genome. Inparticular embodiments, the invention provides an AAV particlecomprising an AAV capsid comprising an AAV capsid protein of theinvention, wherein the AAV capsid packages an AAV vector genome. Theinvention also provides an AAV particle comprising an AAV capsid or AAVcapsid protein encoded by the chimeric nucleic acid capsid codingsequences of the invention.

In particular embodiments, the virion is a recombinant vector comprisinga heterologous nucleic acid of interest, e.g., for delivery to a cell.Thus, the present invention is useful for the delivery of nucleic acidsto cells in vitro, ex vivo, and in vivo. In representative embodiments,the recombinant vector of the invention can be advantageously employedto deliver or transfer nucleic acids to animal (e.g., mammalian) cells.

Any heterologous nucleotide sequence(s) may be delivered by a virusvector of the present invention. Nucleic acids of interest includenucleic acids encoding polypeptides, optionally therapeutic (e.g., formedical or veterinary uses) and/or immunogenic (e.g., for vaccines)polypeptides.

In some embodiments, the polypeptide is one that stimulates growthand/or differentiation of oligodendrocytes. Examples include, withoutlimitation, insulin-like growth factor-1, glial-derived neurotrophicfactor, neurotrophin-3, artemin, transforming growth factor alpha,platelet-derived growth factor, leukemia inhibitory factor, prolactin,monocarboxylate transporter 1, or nuclear factor 1A.

Therapeutic polypeptides include, but are not limited to, thosedescribed above.

Heterologous nucleotide sequences encoding polypeptides include thoseencoding reporter polypeptides as described above.

Alternatively, the heterologous nucleic acid may encode an antisenseoligonucleotide, a ribozyme, RNAs that effect spliceosome-mediatedtrans-splicing, interfering RNAs (RNAi) including small interfering RNAs(siRNA) that mediate gene silencing, microRNA, or other non-translated“functional” RNAs, such as “guide” RNAs, and the like as describedabove.

The present invention also provides recombinant virus vectors thatexpress an immunogenic polypeptide, e.g., for vaccination, as describedabove.

Alternatively, the heterologous nucleotide sequence may encode anypolypeptide that is desirably produced in a cell in vitro, ex vivo, orin vivo. For example, the virus vectors may be introduced into culturedcells and the expressed protein product isolated therefrom.

It will be understood by those skilled in the art that the heterologousnucleic acid(s) of interest may be operably associated with appropriatecontrol sequences as described above. Advantageously, theoligodendrocyte-specific chimeric capsids of the present inventionpermit the use of constitutive promoters to express the heterologousnucleic acid(s) of interest in an oligodendrocyte-specific manner, ascompared to prior art AAV vectors which required the use ofoligodendrocyte-specific promoters.

The invention also provides chimeric AAV particles comprising an AAVcapsid and an AAV genome, wherein the AAV genome “corresponds to” (i.e.,encodes) the AAV capsid. Also provided are collections or libraries ofsuch chimeric AAV particles, wherein the collection or library comprises2 or more, 10 or more, 50 or more, 100 or more, 1000 or more, 10⁴ ormore, 10⁵ or more, or 10⁶ or more distinct sequences.

The present invention further encompasses “empty” capsid particles(i.e., in the absence of a vector genome) comprising, consisting of, orconsisting essentially of the chimeric AAV capsid proteins of theinvention. The chimeric AAV capsids of the invention can be used as“capsid vehicles,” as has been described in U.S. Pat. No. 5,863,541.Molecules that can be covalently linked, bound to or packaged by thevirus capsids and transferred into a cell include DNA, RNA, a lipid, acarbohydrate, a polypeptide, a small organic molecule, or combinationsof the same. Further, molecules can be associated with (e.g., “tetheredto”) the outside of the virus capsid for transfer of the molecules intohost target cells. In one embodiment of the invention the molecule iscovalently linked (i.e., conjugated or chemically coupled) to the capsidproteins. Methods of covalently linking molecules are known by thoseskilled in the art.

The virus capsids of the invention also find use in raising antibodiesagainst the novel capsid structures. As a further alternative, anexogenous amino acid sequence may be inserted into the virus capsid forantigen presentation to a cell, e.g., for administration to a subject toproduce an immune response to the exogenous amino acid sequence.

The invention also provides nucleic acids (e.g., isolated nucleic acids)encoding the chimeric virus capsids and chimeric capsid proteins of theinvention. Further provided are vectors comprising the nucleic acids,and cells (in vivo or in culture) comprising the nucleic acids and/orvectors of the invention. Such nucleic acids, vectors and cells can beused, for example, as reagents (e.g., helper constructs or packagingcells) for the production of virus vectors as described herein.

In exemplary embodiments, the invention provides nucleic acid sequencesencoding the AAV capsid of SEQ ID NOS:130-132 or at least 90% identicalto the nucleotide sequence of SEQ ID NO:129. The invention also providesnucleic acids encoding the AAV capsid variants, capsid protein variantsand fusion proteins as described above. In particular embodiments, thenucleic acid hybridizes to the complement of the nucleic acid sequencesspecifically disclosed herein under standard conditions as known bythose skilled in the art and encodes a variant capsid and/or capsidprotein. Optionally, the variant capsid or capsid protein substantiallyretains at least one property of the capsid and/or capsid or capsidprotein encoded by the nucleic acid sequence of SEQ ID NO:129. Forexample, a virus particle comprising the variant capsid or variantcapsid protein can substantially retain the oligodendrocyte tropismprofile of a virus particle comprising a capsid or capsid proteinencoded by a nucleic acid coding sequence of SEQ ID NO:129.

For example, hybridization of such sequences may be carried out underconditions of reduced stringency, medium stringency or even stringentconditions as described above.

In other embodiments, nucleic acid sequences encoding a variant capsidor capsid protein of the invention have at least about 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity with thenucleic acid sequence of SEQ ID NO:129 and optionally encode a variantcapsid or capsid protein that substantially retains at least oneproperty of the capsid or capsid protein encoded by a nucleic acid ofSEQ ID NO:129.

As is known in the art, a number of different programs can be used toidentify whether a nucleic acid or polypeptide has sequence identity toa known sequence as described above.

In particular embodiments, the nucleic acid can comprise, consistessentially of, or consist of a vector including but not limited to aplasmid, phage, viral vector (e.g., AAV vector, an adenovirus vector, aherpesvirus vector, or a baculovirus vector), bacterial artificialchromosome (BAC), or yeast artificial chromosome (YAC). For example, thenucleic acid can comprise, consist of, or consist essentially of an AAVvector comprising a 5′ and/or 3′ terminal repeat (e.g., 5′ and/or 3′ AAVterminal repeat).

In some embodiments, the nucleic acid encoding the chimeric AAV capsidprotein further comprises an AAV rep coding sequence. For example, thenucleic acid can be a helper construct for producing viral stocks.

The invention also provides packaging cells stably comprising a nucleicacid of the invention as described above.

The nucleic acid can be incorporated into a delivery vector, such as aviral delivery vector. To illustrate, the nucleic acid of the inventioncan be packaged in an AAV particle, an adenovirus particle, aherpesvirus particle, a baculovirus particle, or any other suitablevirus particle.

Moreover, the nucleic acid can be operably associated with a promoterelement. Promoter elements are described in more detail herein.

The present invention further provides methods of producing the virusvectors of the invention as described above.

The inventive packaging methods may be employed to produce high titerstocks of virus particles. In particular embodiments, the virus stockhas a titer of at least about 10⁵ transducing units (tu)/ml, at leastabout 10⁶ tu/ml, at least about 10⁷ tu/ml, at least about 10⁸ tu/ml, atleast about 10⁹ tu/ml, or at least about 10¹⁰ tu/ml.

The novel capsid protein and capsid structures find use in raisingantibodies, for example, for diagnostic or therapeutic uses or as aresearch reagent. Thus, the invention also provides antibodies againstthe novel capsid proteins and capsids of the invention as describedabove.

VI. Methods of Using Chimeric AAV Capsids Targeted to Oligodendrocytes

The present invention also relates to methods for deliveringheterologous nucleotide sequences into oligodendrocytes. The virusvectors of the invention may be employed, e.g., to deliver a nucleotidesequence of interest to an oligodendrocyte in vitro, e.g., to produce apolypeptide or nucleic acid in vitro or for ex vivo gene therapy. Thevectors are additionally useful in a method of delivering a nucleotidesequence to a subject in need thereof, e.g., to express a therapeutic orimmunogenic polypeptide or nucleic acid. In this manner, the polypeptideor nucleic acid may thus be produced in vivo in the subject. The subjectmay be in need of the polypeptide or nucleic acid because the subjecthas a deficiency of the polypeptide, or because the production of thepolypeptide or nucleic acid in the subject may impart some therapeuticeffect, as a method of treatment or otherwise, and as explained furtherbelow.

In particular embodiments, the vectors are useful to express apolypeptide or nucleic acid that provides a beneficial effect tooligodendrocytes, e.g., to promote growth and/or differentiation ofoligodendrocytes. The ability to target vectors to oligodendrocytes maybe particularly useful to treat diseases or disorders involvingoligodendrocyte dysfunction and/or demyelination of neurons. In otherembodiments, the vectors are useful to express a polypeptide or nucleicacid that provides a beneficial effect to cells near theoligodendrocytes (e.g., neurons).

Thus, one aspect of the invention relates to a method of delivering anucleic acid of interest to an oligodendrocyte, the method comprisingcontacting the oligodendrocyte with the AAV particle of the invention.

In another aspect, the invention relates to a method of delivering anucleic acid of interest to an oligodendrocyte in a mammalian subject,the method comprising administering an effective amount of the AAVparticle or pharmaceutical formulation of the invention to a mammaliansubject.

A further aspect of the invention relates to a method of treating adisorder associated with oligodendrocyte dysfunction in a subject inneed thereof, the method comprising administering a therapeuticallyeffective amount of the AAV particle of the invention to the subject. Inone embodiment, the disorder associated with oligodendrocyte dysfunctionis a demyelinating disease. In one embodiment, the disorder associatedwith oligodendrocyte dysfunction is multiple sclerosis,Pelizaeus-Merzbacher disease, Krabbe's disease, metachromaticleukodystrophy, adrenoleukodystrophy, Canavan disease, Alexanderdisease, orthochromatic leukodystrophy, Zellweger disease, 18q-syndrome,cerebral palsy, spinal cord injury, traumatic brain injury, stroke,phenylketonuria, or viral infection, or any other disorder known orlater found to be associated with oligodendrocyte dysfunction. Inanother embodiment, the methods of the invention are used to treat adisorder that is not directly associated with oligodendrocytedysfunction but would benefit by expression of a heterologouspolypeptide or nucleic acid in oligodendrocytes in addition to orinstead of expression in neurons, astrocytes, or other CNS cell types.Examples include, without limitation, neurodegenerative disorders suchas Alzheimer's disease, Parkinson's disease, and Huntington's disease,CNS tumors, and other CNS disorders as described above.

In another aspect of the invention, the chimeric AAV capsid and vectorsof the invention are fully- or nearly fully-detargeted vectors that canbe further modified to a desirable tropic profile for targeting of oneor more peripheral organs or tissues as described above. In this aspect,the present invention also relates to methods for deliveringheterologous nucleotide sequences into a broad range of cells, includingdividing and non-dividing cells. The virus vectors of the invention maybe employed to deliver a nucleotide sequence of interest to a cell invitro, e.g., to produce a polypeptide in vitro or for ex vivo genetherapy. The vectors are additionally useful in a method of delivering anucleotide sequence to a subject in need thereof, e.g., to express atherapeutic or immunogenic polypeptide or nucleic acid. In this manner,the polypeptide or nucleic acid may thus be produced in vivo in thesubject. The subject may be in need of the polypeptide or nucleic acidbecause the subject has a deficiency of the polypeptide, or because theproduction of the polypeptide or nucleic acid in the subject may impartsome therapeutic effect, as a method of treatment or otherwise, and asexplained further below.

In general, the virus vectors of the invention may be employed todeliver any foreign nucleic acid with a biological effect to treat orameliorate the symptoms associated with any disorder related to geneexpression. Further, the invention can be used to treat any diseasestate for which it is beneficial to deliver a therapeutic polypeptide.Illustrative disease states include those described above.

The virus vectors according to the present invention may also beemployed to provide an antisense nucleic acid or inhibitory RNA (e.g.,microRNA or RNAi such as a siRNA or shRNA) to a cell in vitro or invivo. Expression of the inhibitory RNA in the target cell diminishesexpression of a particular protein(s) by the cell. Accordingly,inhibitory RNA may be administered to decrease expression of aparticular protein in a subject in need thereof. Inhibitory RNA may alsobe administered to cells in vitro to regulate cell physiology, e.g., tooptimize cell or tissue culture systems.

As a further aspect, the virus vectors of the present invention may beused to produce an immune response in a subject. According to thisembodiment, a virus vector comprising a nucleic acid encoding animmunogen may be administered to a subject, and an active immuneresponse (optionally, a protective immune response) is mounted by thesubject against the immunogen. Immunogens are as described hereinabove.

Alternatively, the virus vector may be administered to a cell ex vivoand the altered cell is administered to the subject. The heterologousnucleic acid is introduced into the cell, and the cell is administeredto the subject, where the heterologous nucleic acid encoding theimmunogen is optionally expressed and induces an immune response in thesubject against the immunogen. In particular embodiments, the cell is anantigen-presenting cell (e.g., a dendritic cell).

The virus vectors of the present invention may also be administered forcancer immunotherapy by administration of a viral vector expressing acancer cell antigen (or an immunologically similar molecule) or anyother immunogen that produces an immune response against a cancer cellas described above. The virus vector may be administered to a subject invivo or by using ex vivo methods, as described herein.

In particular embodiments, cells may be removed from a subject withcancer and contacted with a virus vector according to the presentinvention. The modified cell is then administered to the subject,whereby an immune response against the cancer cell antigen is elicited.This method is particularly advantageously employed withimmunocompromised subjects that cannot mount a sufficient immuneresponse in vivo (i.e., cannot produce enhancing antibodies insufficient quantities).

It is known in the art that immune responses may be enhanced byimmunomodulatory cytokines (e.g., α-interferon, β-interferon,γ-interferon, ω-interferon, τ-interferon, interleukin-1α,interleukin-1β, interleukin-2, interleukin-3, interleukin-4, interleukin5, interleukin-6, interleukin-7, interleukin-8, interleukin-9,interleukin-10, interleukin-11, interleukin 12, interleukin-13,interleukin-14, interleukin-18, B cell Growth factor, CD40 Ligand, tumornecrosis factor-α, tumor necrosis factor-β, monocyte chemoattractantprotein-1, granulocyte-macrophage colony stimulating factor, andlymphotoxin). Accordingly, immunomodulatory cytokines (e.g., CTLinductive cytokines) may be administered to a subject in conjunctionwith the virus vectors.

Cytokines may be administered by any method known in the art. Exogenouscytokines may be administered to the subject, or alternatively, anucleotide sequence encoding a cytokine may be delivered to the subjectusing a suitable vector, and the cytokine produced in vivo.

The viral vectors are further useful for targeting oligodendrocytes forresearch purposes, e.g., for study of CNS function in vitro or inanimals or for use in creating and/or studying animal models of disease.For example, the vectors can be used to deliver heterologous nucleicacids to oligodendrocytes in animal models of demyelinating diseases.Demyelination can be induced in animals by a variety of means, includingwithout limitation administration of viruses (e.g., Semliki virus,murine hepatitis virus, or Theiler's murine encephalomyelitis virus) andadministration of chemicals (e.g., cuprizone, ethidium bromide, orlysolecithin). In some embodiments, the vector can also be used inanimal models of experimental autoimmune encephalomyelitis. Thiscondition can be induced by, for example, administration of kainite,SIN-1, anti-galactocerebroside, or irradiation. In other embodiments,the viral vector can be used to specifically deliver to oligodendrocytesa toxic agent or an enzyme that produces a toxic agent (e.g., thymidinekinase) in order to kill some or all of the cells.

Further, the virus vectors according to the present invention findfurther use in diagnostic and screening methods, whereby a gene ofinterest is transiently or stably expressed in a cell culture system, oralternatively, a transgenic animal model. The invention can also bepracticed to deliver a nucleic acid for the purposes of proteinproduction, e.g., for laboratory, industrial or commercial purposes.

Recombinant virus vectors according to the present invention find use inboth veterinary and medical applications. Suitable subjects include bothavians and mammals as described above. Optionally, the subject is “inneed of” the methods of the present invention, e.g., because the subjecthas or is believed at risk for a disorder including those describedherein or that would benefit from the delivery of a nucleic acidincluding those described herein. For example, in particularembodiments, the subject has (or has had) or is at risk for ademyelinating disorder or a spinal cord or brain injury. As a furtheroption, the subject can be a laboratory animal and/or an animal model ofdisease.

In particular embodiments, the present invention provides apharmaceutical composition comprising a virus vector of the invention ina pharmaceutically acceptable carrier and, optionally, other medicinalagents, pharmaceutical agents, stabilizing agents, buffers, carriers,adjuvants, diluents, etc. For injection, the carrier will typically be aliquid. For other methods of administration, the carrier may be eithersolid or liquid. For inhalation administration, the carrier will berespirable, and will preferably be in solid or liquid particulate form.

By “pharmaceutically acceptable” it is meant a material that is nottoxic or otherwise undesirable, i.e., the material may be administeredto a subject without causing any undesirable biological effects.

One aspect of the present invention is a method of transferring anucleotide sequence to a cell in vitro. The virus vector may beintroduced to the cells at the appropriate multiplicity of infectionaccording to standard transduction methods appropriate for theparticular target cells. Titers of the virus vector or capsid toadminister can vary, depending upon the target cell type and number, andthe particular virus vector or capsid, and can be determined by those ofskill in the art without undue experimentation. In particularembodiments, at least about 10³ infectious units, more preferably atleast about 10⁵ infectious units are introduced to the cell.

The cell(s) into which the virus vector can be introduced may be of anytype as described above.

The virus vectors may be introduced to cells in vitro for the purpose ofadministering the modified cell to a subject. In particular embodiments,the cells have been removed from a subject, the virus vector isintroduced therein, and the cells are then replaced back into thesubject. Methods of removing cells from subject for treatment ex vivo,followed by introduction back into the subject are known in the art(see, e.g., U.S. Pat. No. 5,399,346). Alternatively, the recombinantvirus vector is introduced into cells from another subject, intocultured cells, or into cells from any other suitable source, and thecells are administered to a subject in need thereof.

Suitable cells for ex vivo gene therapy are as described above. Dosagesof the cells to administer to a subject will vary upon the age,condition and species of the subject, the type of cell, the nucleic acidbeing expressed by the cell, the mode of administration, and the like.Typically, at least about 10² to about 10⁸ or about 10³ to about 10⁶cells will be administered per dose in a pharmaceutically acceptablecarrier. In particular embodiments, the cells transduced with the virusvector are administered to the subject in an effective amount incombination with a pharmaceutical carrier.

In some embodiments, cells that have been transduced with the virusvector may be administered to elicit an immunogenic response against thedelivered polypeptide (e.g., expressed as a transgene or in the capsid)as described above.

A further aspect of the invention is a method of administering the virusvectors or capsids of the invention to subjects. In particularembodiments, the method comprises a method of delivering a nucleic acidof interest to an animal subject, the method comprising: administeringan effective amount of a virus vector according to the invention to ananimal subject. Administration of the virus vectors of the presentinvention to a human subject or an animal in need thereof can be by anymeans known in the art. Optionally, the virus vector is delivered in aneffective dose in a pharmaceutically acceptable carrier.

The virus vectors of the invention can further be administered to asubject to elicit an immunogenic response (e.g., as a vaccine) asdescribed above.

Dosages of the virus vectors to be administered to a subject will dependupon the mode of administration, the disease or condition to be treated,the individual subject's condition, the particular virus vector, and thenucleic acid to be delivered, and can be determined in a routine manner.Exemplary doses for achieving therapeutic effects are virus titers of atleast about 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³, 10¹⁴, 10¹⁵transducing units or more, preferably about 10⁷ or 10⁸, 10⁹, 10¹⁰, 10¹¹,10¹², 10¹³ or 10¹⁴ transducing units, yet more preferably about 10¹²transducing units.

In particular embodiments, more than one administration (e.g., two,three, four or more administrations) may be employed to achieve thedesired level of gene expression over a period of various intervals,e.g., daily, weekly, monthly, yearly, etc.

Exemplary modes of administration include oral, rectal, transmucosal,topical, intranasal, inhalation (e.g., via an aerosol), buccal (e.g.,sublingual), vaginal, intrathecal, intraocular, transdermal, in utero(or in ovo), parenteral (e.g., intravenous, subcutaneous, intradermal,intramuscular [including administration to skeletal, diaphragm and/orcardiac muscle], intradermal, intrapleural, intracerebral, andintraarticular), topical (e.g., to both skin and mucosal surfaces,including airway surfaces, and transdermal administration),intro-lymphatic, and the like, as well as direct tissue or organinjection (e.g., to liver, skeletal muscle, cardiac muscle, diaphragmmuscle or brain). Administration can also be to a tumor (e.g., in or anear a tumor or a lymph node). The most suitable route in any given casewill depend on the nature and severity of the condition being treatedand on the nature of the particular vector that is being used.

In some embodiments, the viral vector is administered directly to theCNS, e.g., the brain or the spinal cord. Direct administration canresult in high specificity of transduction of oligodendrocytes, e.g.,wherein at least 80%, 85%, 90%, 95% or more of the transduced cells areoligodendrocytes. Any method known in the art to administer vectorsdirectly to the CNS can be used. The vector may be introduced into thespinal cord, brainstem (medulla oblongata, pons), midbrain(hypothalamus, thalamus, epithalamus, pituitary gland, substantia nigra,pineal gland), cerebellum, telencephalon (corpus striatum, cerebrumincluding the occipital, temporal, parietal and frontal lobes, cortex,basal ganglia, hippocampus and amygdala), limbic system, neocortex,corpus striatum, cerebrum, and inferior colliculus. The vector may alsobe administered to different regions of the eye such as the retina,cornea or optic nerve. The vector may be delivered into thecerebrospinal fluid (e.g., by lumbar puncture) for more disperseadministration of the vector.

The delivery vector may be administered to the desired region(s) of theCNS by any route known in the art, including but not limited to,intrathecal, intracerebral, intraventricular, intranasal, intra-aural,intra-ocular (e.g., intra-vitreous, sub-retinal, anterior chamber) andperi-ocular (e.g., sub-Tenon's region) delivery or any combinationthereof.

Typically, the viral vector will be administered in a liquid formulationby direct injection (e.g., stereotactic injection) to the desired regionor compartment in the CNS. In some embodiments, the vector can bedelivered via a reservoir and/or pump. In other embodiments, the vectormay be provided by topical application to the desired region or byintra-nasal administration of an aerosol formulation. Administration tothe eye or into the ear, may be by topical application of liquiddroplets. As a further alternative, the vector may be administered as asolid, slow-release formulation. Controlled release of parvovirus andAAV vectors is described by international patent publication WO01/91803.

In some embodiments where the subject has a compromised blood-brainbarrier (BBB), the viral vector can be delivered systemically (e.g.,intravenously) to the subject, wherein the vector transducesoligodendrocytes in the area of (e.g., bordering) the BBB compromise. Incertain embodiments, the vector transduces cells in the compromised areabut not cells in uncompromised areas. Thus, one aspect of the inventionrelates to a method of delivering a nucleic acid of interest to an areaof the CNS bordering a compromised blood brain barrier area in amammalian subject, the method comprising intravenously administering aneffective amount of the AAV particle of the invention.

In some embodiments, the compromise in the BBB is due to a disease ordisorder as described above. In other embodiments, the BBB compromisecan be an induced disruption, e.g., to promote delivery of agents to theCNS as described above.

Injectables can be prepared in conventional forms, either as liquidsolutions or suspensions, solid forms suitable for solution orsuspension in liquid prior to injection, or as emulsions. Alternatively,one may administer the virus vector in a local rather than systemicmanner, for example, in a depot or sustained-release formulation.Further, the virus vector can be delivered dried to a surgicallyimplantable matrix such as a bone graft substitute, a suture, a stent,and the like (e.g., as described in U.S. Pat. No. 7,201,898).

Pharmaceutical compositions suitable for different routes ofadministration can be as described above.

Having described the present invention, the same will be explained ingreater detail in the following examples, which are included herein forillustration purposes only, and which are not intended to be limiting tothe invention.

Example 1 Development of AAV Capsids with Enhanced CNS Tropism andMinimal Peripheral Organ Tropism

The AAV capsids were developed through a process called capsid DNAshuffling and directed evolution, which was used to generate a libraryof novel AAV capsid sequences. These capsids were then subjected tomultiple rounds of selective pressure in mice, with potential additionalcapsid mutagenesis occurring in between rounds of selection. Recoveredcapsid clones were used as vectors to a reporter transgene (GFP) ortherapeutic transgene (MeCP2 for Rett syndrome) and evaluated in micefor biodistribution and therapeutic potential.

The original library used consisted of shuffled capsids from AAVserotypes 1, 2, 6, 8, 9, and rh10. Additional engineered capsids werealso incorporated: AAV2.5, AAV2i8, AAV9.47, Seiz32, Seiz83, andundescribed capsids from Dr. Gray's laboratory (retrograde clones 1 and114). The library was produced as previously described (Li et al., Mol.Ther. 16:1252-1260 (2008)). Either wildtype mice or mice modelling Rettsyndrome (B6.129P2(C)-Mecp2^(tm1.1Bird)/J) were used for in vivoselection. For each round of selection, mice (WT mice, knockout maleRett mice, heterozygous female Rett mice) received a single lumbarintrathecal injection of the library, then 2-5 days later tissue wasrecovered from the cervical spinal cord and multiple regions of thebrain. From these samples, the tissue was mechanically dissociated topreferentially recover neurons as described (Li et al., Mol. Ther.16:1252-1260 (2008)). DNA was recovered from neuron-enriched samplesusing a DNeasy blood and tissue kit (Qiagen, cat. #69506). Error-pronePCR was employed to further diversify the library between rounds, usingtaq polymerase with a low starting template and 50 amplification cycles,with primers previously described (Li et al., Mol. Ther. 16:1252-1260(2008)). The pooled PCR products were cloned back into a WT AAV backbone(pSSV9) and pooled clones were used to generate the next round'sstarting library. Pooled clones were transfected into HEK293 cells withan adenovirus helper plasmid (pXX680) and a 10-fold excess of pXR2containing AAV2 Rep and Cap. By this method, chimeric capsids werepackaged into mostly AAV2 capsids. Then the AAV2-encapsidated chimeraswere added to HEK293 cells at an MOI of 0.5 genomes per cell with WTadenovirus at an MOI of 5 infectious units per cell to predominantlypackage each chimeric AAV genome in its own capsid. After 72 hours thecells were harvested and the virus purified as described (Grieger etal., Nat. Protoc. 1:1412-28 (2006)) and titered by qPCR. A total of 3rounds of selection were performed. A sampling of recovered capsidsafter each round were subcloned into recombinant AAV2 backbones (lackingITR elements) and SSV9 replication-competent backbones and sequenced.

To evaluate the biodistribution and (in some cases therapeuticpotential) of the recovered capsids, some clones were dosed into mice bya single lumbar intrathecal administration in a volume of 5 microliters.When evaluating biodistribution, mice were killed 2-4 weekspost-injection, and tissue samples were analyzed for vector DNAbiodistribution as described (Li et al., Mol. Ther. 16:1252-1260(2008)). When evaluating the therapeutic potential of the recoveredcapsid clones, the human MeCP2 gene (driven by the mouse MeCP2 promoter)was packaged into each capsid, then dosed into male knockout Rett miceat 4-5 weeks of age by lumbar intrathecal injection. Mice were monitoredfor the time at which they lost 20% of their peak body weight, which wasused as a pre-determined endpoint to indicate survival as previouslydescribed (Gadalla et al., Mol. Ther. 21(1):18-30 (2013)).

Representative clones are shown in FIGS. 1-3. The CNS tropism of clonesITbrain-2.02 and ITbrain-2.04 is shown in FIGS. 4A-4E. Except whereindicated in FIG. 4A, adult WT C57BL/6 mice were injected IT with 1×10¹⁰vg of the scAAV/GFP vector, then sacrificed at 3 weeks post-injectionfor IHC of GFP expression and qPCR biodistribution to peripheral organs.FIG. 4B=forebrain, cortex. FIG. 4C=midbrain, hippocampus. FIG.4D=hindbrain, cerebellum. FIG. 4E shows the ventral horn and centralcanal of the lumbar spinal cord. n.d.=no data. Scale bars in (A)indicate S.E.M. Scale bar for (B-E) is shown in the lower right and is100 microns. AAV9 represents a current “gold standard” for widespreadCNS gene transfer after intra-CSF administration, and Olig001 is aseparately-derived shuffled capsid. FIG. 5 shows the CNS and peripheraldistribution of clones ITcord-1.06 and ITcord3.03. Adult WT C57BL/6 micewere injected IT with 1×10¹⁰ vg of the scAAV/GFP vector, then sacrificedat 3 weeks post-injection for qPCR biodistribution to CNS tissues andperipheral organs. Error bars indicate SEM. FIG. 6 shows the CNS tropismof clone RTTF-1.11. The figure shows anti-GFP immunohistochemistry onbrain sections from mice that received an intrathecal injection of cloneF1.11 (packaging the GFP gene, 1×10¹⁰ vg per mouse) at 4-5 weeks of age,then were sacrificed after 4 weeks. Images from the Rett mouse (left)show stronger transduction along the entire rostral-caudal axis,compared to the WT mouse (right). Representative results are shown.

The transduction efficiency and tropism of 9 chimeric AAV/GFP viruseswas tested. Each virus was injected intracisternally (5E10 vg per mouse)into three MeCP2+/− female mice that were 5-7 months old (except whereotherwise noted). Three weeks after injections, mice were perfused andbrains were harvested. After 48 hours of fixation in 1×PBS containing 4%paraformaldehyde, the brains were sectioned at 50 μm using a Leica VT1000S vibrating-blade microtome. Sections were incubated for 1 hour atroom temperature in 5% normal goat serum in 0.3M PBST, then incubated40-48 hours at 4° C. in primary antibody solution (5% goat serum in 0.3MPBST, chicken anti-GFP (Ayes; 1:500) plus rabbit anti-mouse MeCP2 (CellSignaling; 1:500), or chicken anti-GFP (Ayes; 1:500) plus rabbitanti-mouse NeuN (Cell Signaling; 1:500)). After washing three times with0.3M PBST, the sections were incubated for 4 hours at room temperaturein secondary antibody solution (0.3M PBST, goat anti-chicken Alexa-fluor488 (Invitrogen; 1:1000), goat anti-rabbit Alexa-fluor 594 (Invitrogen;1:1000)), then washed three more times in 0.3M PBST. Sections were thenincubated with 0.5 pg/mL DAPI in 0.3M PBST for 30 minutes at roomtemperature and washed once with 0.3M PBS. Immuno-labeled sections wereimaged using a Zeiss LSM 780 confocal microscope. Images were takenusing a 20× objective with 4× digital zoom.

To estimate the transduction efficiencies for specific brain regions,the ratio of GFP-expressing neurons to DAPI-stained nuclei wascalculated for random fields (n=12-25) from sections of hippocampus,cortex, brainstem, subiculum, cerebral nuclei, and cerebellum. The meantransduction efficiency per capsid was calculated by averaging the meanefficiencies across all analyses (FIG. 7). To determine MeCP2 tropism,the ratio of GFP+/MeCP2+ neurons to GFP+ neurons was calculated for eachof the following regions: hippocampus, cortex, brainstem, subiculum,cerebral nuclei, and cerebellum (FIG. 8). Cells with glial morphologywere not used to calculate MeCP2 tropism as genetically WT glial cellsmay express MeCP2 at levels that are too low for detection byimmunofluorescence. Similar methods were used to calculate NeuN tropismas was done for MeCP2 (FIG. 9).

Example 2 Development of AAV Capsids that Preferentially TargetOligodendrocytes Materials and Methods AAV Capsid DNA Shuffling and InVivo Clone Rescue

A library consisting of shuffled capsids from AAV serotypes AAVserotypes 1-6, 8, 9, rh10, several chimeric capsids and mutant capsids,and an AAV8 with an E533K mutation was produced using methods aspreviously described (Li et al., Mol. Ther. 16:1252 (2008)). Theshuffled library was injected intravenously into rats that previouslyreceived a striatal 6-hydroxy-dopamine treatment. Three days later, therats were killed and cells were mechanically dissociated from striatum.DNA was recovered from neuron-enriched samples using the Qiagen DNeasyblood and tissue kit and subsequently concentrated by ethanolprecipitation. The Expand Long Template PCR System (cat. no.11681834001, low starting template, 50 cycles; Roche, Indianapolis,Ind.) was used to recover the intact capsid library sequences, withprimers previously described (Li et al., Mol. Ther. 16:1252 (2008)). Asubsequent error-prone PCR step was employed to further diversify thelibrary between rounds. The pooled mutagenized PCR products were clonedback into a WT AAV backbone (pSSV9) and pooled clones were used togenerate the next round's starting library. Pooled clones weretransfected into HEK293 cells with an adenovirus helper plasmid (pXX680)and a 10-fold excess of pXR2 containing AAV2 rep and cap. By thismethod, chimeric capsid genomes were packaged into mostly AAV2 capsids.The titer of the AAV2-encapsidated chimeric library was determined usingqPCR. Then, the AAV2-encapsidated chimeras were added to HEK293 cells ata multiplicity of infection of 0.5 vg/cell with WT adenovirus at amultiplicity of infection of 5 infectious units per cell topredominantly package each chimeric AAV genome in its own capsid. After48 h, the cells were harvested and the virus purified as described (Grayet al., Gene Ther. 20:450 (2013)) and titered by qPCR. A total of tworounds of selection were performed. Recovered clones were recoveredafter each round and subcloned into rAAV pXR2 backbones and SSV9replication-competent backbones and sequenced.

Cloning

AAV8/E532K was made to introduce the single mutation (E532K, usingOlig001 VP1 numbering) in pGSK2/8 (repAAV2-capAAV8) using site-directedmutagenesis (Agilent quik change II kit). Primers were designed usingthe Agilent QuikChange Primer Design Program; forward:5′GGGAAAAAAACGCTCCTTGTCGTCTTTGTGTGTTG3′ (SEQ ID NO:135) and reverse:5′CAACACACAAAGA CGACAAGGAGCGTTTTTTTCCC (SEQ ID NO:136). Single colonieswere grown up and verified by Sanger sequencing. To make Olig001/AAV8VP3, the N-terminal of Olig001 containing VP1 and VP2 was amplifiedusing forward (F1): 5′AATGTGGATTTGGATGACTG (SEQ ID NO:137) and amutagenic reverse primer at the VP3 transcription start:5′CGTTATTGTCTGCCATTGGTGCGCCACCGCCTGCAGCCATTGTAAGAGA3′ (SEQ ID NO:138)resulting in a 659 bp fragment. The C-terminal portion of AAV8 VP3sequence was amplified from pGSK2/8 using the forward primer used:5′ACCAATGGCAGACAATAACGAAG GCGCCGACGGAGTGGGTA3′ (SEQ ID NO:139) andreverse primer used (R2): 5′AGAGCCGAGAACGTAC3′ (SEQ ID NO:140) resultingin a 437 bp product. The two PCR products had 20 bp overlapping sequencewith each other. The full chimeric cap gene was amplified using bothfragments and F1 and R2 primers. The final PCR product (Olig001/AAV8VP3) and pGSK2/8 was digested with SwaI and BsiWI. The 6266 bp GSK2/8band and 1070 bp chimeric cap gene PCR product were gel extracted.Fragments were ligated at a 3:1 insert to vector molar ratio using 100ng of pGSK2/8 vector. Ligation mixes were transformed into Blue-XL(Agilent; 200249) cells and plated onto LB-Amp plates. Single colonieswere grown up and verified via Sanger sequencing.

AAV Vector Production

Recombinant AAVs were produced using a triple plasmid transfectionmethod in HEK293 cells, follow by iodixanol gradient centrifugation andion-exchange chromatography, as previously described (Gray et al., GeneTher. 20:450 (2013)). All AAV vectors were packaged with aself-complementary genome with enhanced GFP under the control of a CMVenhancer, miniature chicken beta actin promoter (CBh), and MVM intron(Gray et al., Hum. Gene Ther. 22:1143 (2011)). Peak fractions weredialyzed in phosphate-buffered saline (PBS) with 5% sorbitol, and NaCladded to a final concentration of 350 mM NaCl. Viral titers wereobtained via qPCR (see below).

qPCR for Biodistribution Studies and Viral Titer

qPCR was used to determine viral titer and for biodistribution studies(Gray et al., Current protocols in neuroscience/editorial board,Jacqueline N. Crawley . . . [et al.] Chapter 4:Unit 4 17 (2011)). Allreactions were done using the SyBR Green-based Lightcycler fast startDNA master mix (Roche) on a Roche 480 Lightcycler instrument, followingthe manufacturer's instructions. To prepare virus samples for titer,they were treated with DNase I for 1 h, then the DNase I was inactivatedwith the addition of EDTA and heating at 70° C. for 10 min. To liberatethe encapsidated viral genomes for qPCR analysis, the reaction mixtureswere digested with Proteinase K for at least 2 h at 50° C., then boiledfor 10 min to inactivate the Proteinase K. Samples were diluted inPCR-grade water and used as template for qPCR reactions. For GFP virusquantification, plasmid DNA was used as the standard. For quantificationof mouse genomic DNA, purified, and quantified mouse genomic DNA wasused as a standard. All successful reactions gave a single product bymelting curve analysis, used a standard curve with an R2 value of 1, andhad a reaction run in parallel containing no template that gave noproduct. GFP primers were as follows:

(SEQ ID NO: 141) Forward: 5′AGCAGCACGACTTCTTCAACTCC3′ (SEQ ID NO: 142)Reverse: 5′TGTAGTTGTACTCCAGCTTGTGCC3′.LaminB2 primers for quantification of mouse genomic DNA were as follows:

(SEQ ID NO: 143) Forward: 5′GTTAACACTCAGGCGCATGGGCC3′ (SEQ ID NO: 144)Reverse: 5′CCAT CAGGGTCACCTCTGGTTCC3′.To titer the AAV vectors, the following primers were used:

(SEQ ID NO: 145) Forward: 5′AACATGCTACGCAGAGAGGGAGTGG3′ (SEQ ID NO: 146)Reverse: 5′CATGAGACAAGGAACCCCTAGTGATGGAG3′.

Animal Procedures

All animals used in these studies were either male Sprague-Dawley rats(Charles River, Morrisville, N.C., USA, 250-250 grams) or adult femaleC57Bl/6 mice (Jackson Labs, Bar Harbor, Me.) that were maintained in a12-h light-dark cycle and had free access to water and food. All careand procedures were in accordance with the National Institutes of HealthGuide for the Care and Use of Laboratory Animals, and all proceduresreceived prior approval by the University of North CarolinaInstitutional Animal Care and Use Committee.

6-Hydroxy-Dopamine Treatment

Initially, rats (N=2) were anesthetized with 50 mg/kg pentobarbital,i.p. and placed into a stereotactic frame. Next the rats received aunilateral infusion of 6-hydroxy-dopamine (2 μl, 20 μg) into the rightstriatum (0.5 mm anterior to bregma, 3.5 mm lateral, 5.5 mm vertical,according to the atlas of Paxinos and Watson (Paxinos G, Watson C., Therat brain in stereotaxic coordinates, 6th ed. Academic Press/Elsevier,Amsterdam; Boston (2007)). This treatment results in a significantreduction in striatal dopamine content 14 days post-treatment.

AAV Capsid Library Administration

The AAV capsid library was administered 14 days post-6-hydroxy-dopaminetreatment. For each selection round 2 rats initially were anesthetizedwith pentobarbital (50 mg/kg i.p.) and subsequently received anintravenous tail vein injection of the AAV capsid library virus. Threedays later, the rats were euthanized and the right striatum wasdissected out. Subsequently, cells were mechanically dissociated aspreviously described (Gray et al., Mol. Ther. 18:570 (2010)) forsubsequent PCR clone rescue.

Stereotactic AAV Vector Administration

As above, rats were anesthetized with pentobarbital and placed into astereotactic frame. Each of the different AAV clones were inphosphate-buffered saline (PBS), 5% sorbitol and 350 mM NaCl) wereinfused at a rate of 1 μl/5 min) into the striatum (0.5 mm anterior tobregma, 3.5 mm lateral, 5.5 mm vertical, according to the atlas ofPaxinos and Watson (Paxinos G, Watson C., The rat brain in stereotaxiccoordinates, 6th ed. Academic Press/Elsevier, Amsterdam; Boston (2007)).The rats were sacrificed for immunohistochemical evaluation 14 dayspost-vector infusion.

Immunohistochemistry

Fourteen days post-vector infusion, rats received an overdose ofpentobarbital (100 mg/kg, i.p.) and subsequently were perfusedtranscardially with 100 ml of ice-cold 0.1 M PBS pH 7.4 (25 ml/min)followed by 180 ml of ice-cold 4% paraformaldehyde-phosphate buffer (pH7.4)(30 ml/min). Each brain was post-fixed in 4%paraformaldehyde-phosphate buffer (pH 7.4) overnight at 4° C. Fixedbrains were sectioned coronally on a Leica vibratome (40-μm thick) andstored in ice-cold 0.1 M PBS pH 7.4 until further processing. Forimmunostaining, slides were washed three times for 5 min in 0.1 M PBS pH7.4, then blocked in 10% goat serum and 0.1% Triton-X in 0.1 M PBS pH7.4 for 30 min. Primary antibodies NeuN (1:500; Millipore; MAB377) andGFAP (1:2000; Dako; Z0334) were incubated in 5% goat serum and 0.05%Triton-X in 0.1 M PBS pH 7.4 at 4° C. with gentle agitation overnight.Sections were washed in 0.1 M PBS pH 7.4 and blocked again as describedabove. Secondary goat anti-mouse Alexa 594 (A11032) for NeuN or goatanti-rabbit Alexa 594 (A11080) for GFAP (both 1:500 in 5% goat serum and0.5% Triton-X in 0.1 M PBS pH 7.4 with gentle agitation at 4° C. for 45min). Sections were washed with 0.1 M PBS pH 7.4 three times for 5 mineach. Sections were floated and put on glass slides and dried overnightat room temperature. Slides were mounted with fluorescent mounting mediaand cover slipped. Confocal imaging was performed at the Michael HookerMicroscopy Core at UNC-Chapel Hill using a Leica Sp2 confocal. Slideswere visualized using the 40× objective using sequential laser scanningto obtain z-stacks. Z-stacks were approximately 4 μm with 10-12 slices0.36 μm thick per stack. Stacks were flattened in the Leica software andprocessed in Image J. At least five independent fields were used tocount GFP positive cells and their co-labeling with NeuN or GFAP todetermine tropism.

Biodistribution

Adult female C57Bl/6 mice (Jackson labs; Bar Harbor, Me.) wereintravenously injected in the tail vein with 5×10¹⁰ vg (˜2.5×10¹² vg/kgbody weight) in 200 μl PBS with 5% D-sorbitol. Ten days post injectionorgans were harvested. Total DNA from each organ was extracted withQiagen DNeasy blood and tissue kit and total copies of GFP and mousegenomic LaminB2 were determined by qPCR. Data was compiled from 5 micefor AAV8 and 4 mice for Olig001.

In Vitro Binding

Mixed glia cultures were prepared from C57BL/6J day three neonatal pups.Forebrains were minced, dissociated, and washed prior to plating in T75flasks. Cells were removed from the flasks and then replated into five35-mm tissue culture dishes with approximately 5×10⁵ cells. At 95%confluency four AAV viruses containing the CBh-GFP reporter genome(Olig001/AAV8 VP3; Olig001; AAV8; AAV8/E532K) were diluted and addedseparately, in quadruplicate, at a MOI of 100 vg/cell and were incubatedat 4° C. with mixing every 10 min for 1 h. Plates were rinsed with icecold PBS 3 times, cells scraped from the plates, pelleted, and frozen at−80° C. A PBS only dish was also included as a mock sample. DNA wasisolated from the samples using the Qiagen DNeasy blood and tissue kit.The amount of viral GFP and mouse genomic LaminB2 was determined byqPCR. Statistical analysis and graphing was done in Prism. Outliers weredetermined using the Grubbs test and subsequently removed. Statisticalsignificance (P<0.05) was determined using a one-tailed Mann-Whitneytest. The fold change was determined from the average of each virus fromAAV8.

Results Identification of an Oligodendrocyte Preferring AAV Capsid

A shuffled AAV capsid library was administered intravenously 2 weeksafter the unilateral administration of 6-hydroxy-dopamine (6-OHDA), and3 days later AAV clones were recovered by PCR from dissociated striatalcells. Surprisingly, 10 of 10 selected clones had highly similar, if notidentical sequences. Even with a second round of capsid shuffling,library administration and clone selection, 12 out of 12 clones hadalmost identical sequences, similar to the first round (FIG. 10A). Whenthe chimeric virus (named Olig001) was administered intravenously tounilateral 6-OHDA treated rats, 2 weeks later immunohistochemistryrevealed only a few GFP positive neurons in the ipsilateral striatum anda sparse number of oligodendrocyte like cells. In marked contrast, 2weeks after a striatal infusion of the Olig001 clone into naïve rats,oligodendrocytes comprised the vast majority of the transduced cells,even though the gene expression was driven by the constitutive CBhpromoter (FIGS. 10B-10E). GFP positive cells exhibited the typicaloligodendrocyte morphology with clear labeling of myelin in the striatalpatch/matrix (FIGS. 10B-10C). Furthermore, GFP positive cells did notco-localize with glial fibrillary acidic protein (GFAP), an astrocytemarker (FIG. 10D), and approximately only 5% of the GFP positive cellsco-localized with NeuN, a neuronal marker (FIG. 10E). Thus, almost allof the GFP positive cells (>95%) were oligodendrocytes with only a fewneurons and no GFP positive astrocytes or microglia. This change intropism directly contrasts the neuronal tropism characteristic of AAV8which shares 99.3% homology (7 amino acid differences) with theVP3-specific portion of the Olig001 capsid sequence (FIG. 10A).

Olig001 is Detargeted from Peripheral Tissues

Given that the selection process involved intravenous administration ofthe capsid library, we sought to characterize the biodistribution ofOlig001 in wild type rodents compared to AAV8. Adult female C57Bl/6 micereceived intravenous administration of equal amounts of either virus.Ten days later, organs were harvested, and the biodistribution wasquantified by qPCR for Olig001-CBh-GFP (white bars) and AAV8-CBh-GFP(gray bars) (FIG. 11). The biodistribution of Olig001 was significantlyreduced in all peripheral organs tested compared to AAV8, especially theliver (FIG. 11). Together with our previous results, these data indicatethat Olig001 has a highly divergent tropism from the related AAV8, bothwithin and outside the CNS.

E532K Mutation in AAV8 Produces a Tropism Switch from Neurons toOligodendrocytes

Of the 7 amino acids that distinguish Olig001 from the VP3 region ofAAV8, only 1 residue (E532K using Olig001 VP1 numbering or E533K usingAAV8 VP1 numbering) has been previously associated with alterations inreceptor/ligand interactions (Wu et al., J. Virol. 80:11393 (2006)). Wuand colleagues discovered that the difference in tissue tropism andligand binding seen between closely related AAV1 and AAV6 is solelyexplained by a lysine or glutamate at the corresponding 532 residues andthat the E533K mutation in AAV8 conferred a new ability to bind heparinsulfate (Wu et al., J. Virol. 80:11393 (2006)). Subsequently, we testedthe influence of the E532K mutation on the tropism of AAV8 in the brain(FIG. 12A). AAV8/E532K was packaged with CBh-GFP and injected at a titerof 2×10⁸ vg/μl into the striatum of wild-type male Sprague-Dawley rats.Two weeks post injection the brains were harvested and assessed fornative GFP co-localization with neuronal (NeuN) and astrocyte (GFAP)markers (FIGS. 12B-12G). Native GFP did not co-localize with eitherneuronal (FIGS. 12B-12D) or astrocyte (FIGS. 12E-12G) markers. Incontrast, GFP positive cells exhibited the characteristic morphology ofoligodendrocytes. Together, AAV8 with the E532K mutation changes thetropism of AAV8 from neuron-preferring to oligodendrocyte-preferring,suggesting a role for the same residue in Olig001 to directoligodendrocyte tropism. However, in the context of Olig001, we foundthat reversing the mutation (K532E) did not affect the oligodendrocytetropism (summarized in FIG. 15).

Olig001 Oligodendrocyte Tropism is Conferred by Amino Acids Outside ofVP3

Traditionally, only VP3 residues are thought to contribute toextracellular receptor binding and the tropism of AAV serotypes, whilespecific VP1 and VP2 portions of the N-terminal portions mediateendosomal escape and nuclear import (Bleker et al., J. Virol. 79:2528(2005); Grieger et al., J. Virol. 80:5199 (2006); Kronenberg et al., J.Virol. 79:5296 (2005); Sonntag et al., J. Virol. 80:11040 (2006)). Toidentify the specific amino acids of the Olig001 capsid that contributeto oligodendrocyte tropism, we made Olig001 mutants that return the 7residue differences within VP3 to AAV8 residues. However, no single orcluster of mutation(s) within Olig001 VP3 decreased the oligodendrocytetropism of Olig001 in the rat striatum to less than 98% of cells(summarized in FIG. 15). Similarly, the peripheral organ biodistributionwas highly reduced in all the tested mutants, with the greatestreduction seen in the mutants that retained the VP1/VP2-specific regionOlig001 (FIG. 16). As a next step, we replaced all of the Olig001 VP3sequence with AAV8 VP3 sequence to assess the overall contribution ofVP3 (FIG. 13A). Mutant Olig001 with an AAV8 VP3 (Olig001/AAV8 VP3) waspackaged with CBh-GFP and injected into the striatum of wild-type maleSprague-Dawley rats. Two weeks later, the brains were harvested andassessed for native GFP co-localization with neuronal (NeuN) andastrocyte (GFAP) markers (FIGS. 13B-13G). Native GFP rarely (in 2% ofcells) co-localized with NeuN (FIGS. 13B-13D) and did not co-localizewith GFAP (FIGS. 13E-13G). Additionally, the GFP positive cellsexhibited the characteristic morphology of striatal oligodendrocytes.These results suggest that VP1/VP2-specific portion of the capsid have apreviously unappreciated influence on AAV tropism.

In Vitro Binding Data

The VP1/VP2-specific portions of the Olig001 capsid could be directingthe preferred transduction of oligodendrocytes through increasedextracellular binding to receptor(s) on oligodendrocytes, or viaenhanced intracellular trafficking by an unknown mechanism that could bespecific to oligodendrocytes. To distinguish between these 2 scenarios,we performed an in vitro binding experiment using mixed glia cultures,including Olig001, the Olig001/VP3 AAV8 mutant, AAV8, and the AAV8/E532Kmutant all packaged with CBh-GFP. Mixed glia cultures were incubatedwith an equivalent amount of each virus for 1 h at 4° C. This procedureallows vector binding to the cell surface, but prevents internalization(Xiao et al., Mol. Ther. 20:317 (2012)). The amount of vector bound tocells was quantified by qPCR for GFP and normalized to mouse genomicLaminB2 (FIG. 14A). Consistent with our in vivo results, Olig001 boundto the mixed glial cell population 9-fold more than AAV8 (FIG. 14B). Thebinding of Olig001/AAV VP3 was slightly lower than Olig001 (4-fold overAAV8), while AAV8/E532K bound 46-fold more than AAV8 (FIG. 14B). Theseresults suggest that both the E532K mutation and the VP1/VP2-specificN-terminal domain each contribute to the oligodendrocyte tropism ofOlig001 in a redundant fashion. Therefore, the in vitro data agrees withthe observed in vivo tropism data.

These studies generated a novel chimeric AAV capsid variant with apreferential in vivo tropism for oligodendrocytes, a finding thatrepresents a major departure from the normal neuronal tropism of thechimera's parental AAV serotypes. Past studies have described theability of AAV2 or AAV8 to transduce oligodendrocytes at a lowefficiency, but these studies required the use of oligodendrocytepromoters to prevent expression in neurons, the preferred cell type forthese vectors (Chen et al., J. Neurosci. Res. 55:504 (1999); Chen etal., Gene Ther. 5:50 (1998); Klein et al., Mol. Ther. 13:517 (2006);Lawlor et al., Mol. Ther. 17:1692 (2009)). In contrast, the Olig001 hasthe ability to efficiently and preferentially transduce oligodendrocytesfollowing intracranial administration, using a ubiquitous promoter.Thus, Olig001 has a preferred tropism for oligodendrocytes and lowtropism for neurons, which is distinct from any previously reported AAVcapsid.

We identified two separate and redundant regions of the Olig001 capsidthat are sufficient to drive this oligodendrocyte tropism.Interestingly, mutation of a single amino acid, E532K, in AAV8(AAV8/E532K) is sufficient to strongly reduce its neuronal tropism infavor of a gained tropism for oligodendrocytes. However, thisoligodendrocyte preference does not arise from a simple loss of neuronaltropism, because the AAV8/E532K mutant shows significant increasedbinding (46-fold over AAV8) to oligodendrocytes in vitro (FIG. 15). Thesecond domain of Olig001 that confers its oligodendrocyte tropism isperhaps more interesting, given its location within the VP1/VP2-specificN-terminus of the capsid ORF. The VP3-specific region of the capsid(which accounts for 54 of the 60 total capsid subunits per virion(Johnson et al., J. Virol. 8:860 (1971); Rose et al., J. Virol. 8:766(1971)) is generally thought to contain the major elements involved withreceptor binding (reviewed in (Agbandje-McKenna et al., Meth. Mol. Biol.807:47 (2011)). In marked contrast, the VP1/VP2 dependent shift in AAVtropism demonstrates that manipulation of the VP3 sequence is not theonly contributing factor to AAV vector tropism. Clearly, our findingsindicate that VP1 and VP2 (which accounts for 54 of the 60 total capsidsubunits per virion (Johnson et al., J. Virol. 8:860 (1971); Rose etal., J. Virol. 8:766 (1971)) can exert a major influence on AAV vectortropism which arises from the extracellular binding rather thanintracellular trafficking.

The Olig001 vector described herein could be useful for in vivo and invitro research applications requiring gene transfer to oligodendrocytes,which are typically refractory to efficient chemical transfection orvector-mediated transduction. Moreover, the ability to efficientlytarget oligodendrocytes in vivo could advance therapeutic strategies fordemyelinating diseases such as Canavan Disease or Krabbe Disease. Thesestudies further challenge the generally accepted notion that the tropismof AAV is dictated solely by the VP3-specific portion of the capsidrather than the VP1/VP1-specific N-terminal domains.

The foregoing is illustrative of the present invention, and is not to beconstrued as limiting thereof. The invention is defined by the followingclaims, with equivalents of the claims to be included therein.

1-171. (canceled)
 172. An AAV capsid protein comprising a polypeptidehaving the sequence of SEQ ID NO: 53, wherein up to 3 amino acids ofsaid polypeptide are substituted, and wherein the substitutions are inthe VP3 portion of the AAV capsid protein.
 173. A nucleic acid encodingthe AAV capsid protein of claim
 172. 174. The nucleic acid of claim 173,wherein the nucleic acid sequence is at least 99% identical to thenucleotide sequence of SEQ ID NO:
 10. 175. The nucleic acid of claim173, wherein the VP1/VP2 portion of the nucleotide sequence is 100%identical to the VP1/VP2 portion of SEQ ID NO:10.
 176. A vectorcomprising the nucleic acid of claim
 173. 177. An AAV particlecomprising the AAV capsid protein of claim 172 and an AAV vector genome,wherein the AAV vector genome comprises a heterologous nucleic acid.178. The AAV particle of claim 177, wherein the heterologous nucleicacid encodes a polypeptide.
 179. The AAV particle of claim 177, whereinthe heterologous nucleic acid encodes an siRNA, a guide RNA, or amicroRNA.
 180. The AAV particle of claim 177, wherein the heterologousnucleic acid is operably linked to an inducible promoter.
 181. The AAVparticle of claim 177, wherein the heterologous nucleic acid is operablylinked to a constitutive promoter.
 182. A method of delivering a nucleicacid of interest to a cell, the method comprising contacting the cellwith an AAV particle of claim
 177. 183. A method of delivering a nucleicacid of interest to a cell in a mammalian subject, the method comprisingadministering to a mammalian subject an effective amount of an AAVparticle of claim
 177. 184. A pharmaceutical formulation comprising theAAV particle of claim 177 and a pharmaceutically acceptable carrier.